Mechanisms underlying the inverse control of parathyroid hormone secretion by calcium.
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Animals↗
Biomedical subjects
M S LeBoff
Publications and source records attributed to M S LeBoff.
Relationship between the concentration and rate of change of calcium and serum intact parathyroid hormone levels in normal humans.
We employed variable rates of infusion of EDTA or calcium gluconate to examine the relationships between the concentration and rate of change of serum total and plasma ionized calcium and serum immunoreactive intact PTH concentrations in normal subjects. Use of a sensitive immunoradiometric assay specific for PTH-(1-84) made it possible to determine the full range of PTH responses to perturbations in the extracellular calcium concentration. By progressively increasing the rate of administration of calcium gluconate or EDTA during rapid or slow infusions, linear rates of change in the serum calcium concentration were achieved in eight men. The slopes were 2-fold greater during the rapid infusions. Both the calcium infusions decreased serum PTH-(1-84) levels from a mean of 23.2 +/- 2.0 (+/- SE) to 6.4 +/- 1.0 and 5.6 +/- 1.0 ng/L for the rapid and slow infusions, respectively, with no obvious rate dependence. The rapid or slow EDTA infusions increased serum PTH levels to the same maximal extent (95.0 +/- 20.2 and 99.9 +/- 14.5 ng/L, respectively), also with no significant rate dependence. Thus, there was no effect of the rate of change of calcium on the PTH response, which was reflected in similar inverse sigmoidal relationships between PTH and serum total and plasma ionized calcium when the data for the slow and rapid infusions were pooled separately. Similar sigmoidal curves were found in normal women. These data suggest the feasibility of using calcium and EDTA infusions combined with an intact PTH assay to define the relationships between circulating levels of PTH-(1-84) and calcium in states of normal and deranged parathyroid physiology.
Adult↗
Secretory control in normal and abnormal parathyroid tissue.
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Animals↗
Permeabilization reveals classical stimulus-secretion coupling in bovine parathyroid cells.
Unlike most secretory cells, high extracellular Ca2+ inhibits, rather than stimulates, PTH release from parathyroid cells. To gain more direct access to the potentially unique secretory apparatus of this cell type, we developed techniques for permeabilizing parathyroid cells using electroshock. Exposure of dispersed bovine parathyroid cells to five 2-kV discharges results in more than 90% uptake of trypan blue and rapid loss of intracellular 86Rb, documenting permeabilization of the plasma membrane. Unlike intact parathyroid cells, a high Ca2+ concentration increases PTH release 2.8-fold from permeabilized cells, with half-maximal and maximal stimulation at 10(-5) and 4 X 10(-4) M Ca2+, respectively, a pattern similar to classical stimulus-secretion coupling. Moreover, PTH release was low at Ca2+ concentrations equivalent to those in intact cells at low extracellular Ca2+ levels (approximately 2 X 10(-7) M). Cellular mechanisms other than changes in the cytosolic Ca2+ concentration per se, therefore, presumably mediate low Ca2+-stimulated PTH release in the intact cell. Diacylglycerol and protein kinase C may play a role in this process, since dioctanoylglycerol (10(-4) M) and 12-O-tetradecanoyl phorbol 13-acetate (10(-6) M) each enhance PTH release from permeabilized cells 2- to 4-fold at a Ca2+ concentration (approximately 2 X 10(-7) M) equivalent to that present in the intact cell at low extracellular Ca2+ concentrations.
Animals↗
Role of cytosolic calcium in the control of cAMP content by calcium in bovine parathyroid cells.
In the present studies we used the calcium (Ca2+)-sensitive dye Quin-2 to determine whether the cytosolic (Cyt) Ca2+ mediates the effects of extracellular (EC) Ca2+ on cAMP accumulation through changes in adenylate cyclase and phosphodiesterase activity in bovine parathyroid cells (bPTC). In dispersed (d) bPTC, increasing the EC Ca2+ from 0.5 to 2 mM produces a rise in the Cyt Ca2+ from 179 to 646 nM which is associated with a 52% inhibition of agonist-stimulated cAMP accumulation. Over this range of free Ca2+ adenylate cyclase activity decreased by approx. half (57%) and phosphodiesterase activity increases 2-fold (101%) suggesting that changes in the Cyt Ca2+ can account for the effects of EC Ca2+ on cAMP through changes in these enzymes. Unlike dbPTC, 4-day-old cultured bPTC show only a 23% suppression of cAMP by high EC Ca2+ and a reduced rise in the Cyt Ca2+ from 0.5 to 3 mM EC Ca2+. Although there is no reduction in the Ca2+ sensitivity of adenylate cyclase, phosphodiesterase activity shows no change at varied free Ca2+. Thus, this diminished Ca2+ sensitivity of phosphodiesterase activity, and the decreased rise in Cyt Ca2+ relative to EC Ca2+ may both contribute to the resistance to the effects of EC Ca2+ on cAMP content in cultured cells. Because in addition to Cyt Ca2+, protein kinase C may also mediate the effects of EC Ca2+ on PTH release, we studied the effects of TPA (12-alpha-tetradecanoylphorbol 13-acetate) on agonist-stimulated cAMP in dbPTC.(ABSTRACT TRUNCATED AT 250 WORDS)
3',5'-Cyclic-AMP Phosphodiesterases↗
Regulation of parathyroid hormone release and cytosolic calcium by extracellular calcium in dispersed and cultured bovine and pathological human parathyroid cells.
Alterations in parathyroid glandular sensitivity to calcium may contribute to the hypersecretion of PTH in hyperparathyroidism. Since the cytosolic calcium concentration may mediate the effects of extracellular calcium on PTH release, we have employed the calcium-sensitive intracellular dye QUIN-2 to examine the relationship between extracellular calcium, cytosolic calcium, and PTH secretion in adult, neonatal, and cultured bovine as well as pathological human parathyroid cells. PTH release was measured using C- and N-terminal radioimmunoassays. Neonatal bovine parathyroid cells showed a greater set-point for secretion (the Ca++ concentration causing half of the maximal inhibition of PTH release) than adult cells (1.27 +/- 0.11 vs. 1.06 +/- 0.11 mM extracellular calcium, P less than 0.01), and a slightly higher extracellular calcium was necessary to raise the cytosolic calcium concentration to a given level in neonatal than in adult bovine parathyroid cells. In individual neonatal and adult cell preparations, there was a close correlation between the set-point for secretion and the "set-point" for cytosolic calcium (r = 0.832, P less than 0.001). In cells from five human parathyroid adenomas, which had an increase in set-point for secretion, the extracellular calcium concentration necessary to raise the cytosolic calcium concentration to a given level was slightly greater than in the neonatal cells. In four preparations of human parathyroid cells there was a significant correlation between the set-points for secretion and cytosolic calcium (r = 0.856, P less than 0.01). Because neonatal bovine and pathological human parathyroid glands show cellular hyperplasia, we studied the temporal relationship between cellular proliferation and the regulation of PTH release and cytosolic calcium concentration in cultured bovine parathyroid cells. Cellular proliferation, estimated by 3H-thymidine incorporation, increased significantly in culture from 104 +/- 10.1 counts/well on day 1 (first 24 h in culture) to 588 +/- 188 and 6,156 +/- 649 counts/well on days 2 and 4, respectively. In cultured cells on day 1, highly Ca++ (2-3 mM) inhibited maximal PTH release by 58.8 +/- 3.2%, which decreased significantly (P less than 0.001) to 38.2 +/- 1.9 and 17.1 +/- 3.7% on days 2 and 4, respectively. The cytosolic calcium observed at 3 mM calcium on day 1 was 701 +/- 43 nM, which declined to 466 +/- 60 and 314 +/- 14 nM on days 2 and 4 (P less than 0.05). There was a close correlation between this progressive decrease in maximal inhibition of PTH release and the cytosolic calcium at high extracellular calcium in cultured cells (r = 0.99, P < 0.001). Thus, during active proliferation of cultured cells, there is an alteration in the regulation of cytosolic calcium at a given extracellular calcium concentration, and changes in the regulation of PTH release and cytosolic calcium by extracellular calcium may be related to enhanced cellular proliferation.
Animals↗
Captopril enhances vascular and adrenal responsiveness to angiotensin II in essential hypertension.
The converting-enzyme inhibitor captopril (25-50 mg orally every 6 h for 66 h) was used to dissociate the circulating levels of angiotensin II (ANG II) from changes in sodium balance in 11 patients with normal renin essential hypertension on 10 mmol of sodium/day intake. Pressor, renal vascular and adrenal responses to graded infusions of ANG II (0.3, 1 and 3 pmol kg-1 min-1) were measured before and after captopril administration. Systemic vascular responses were assessed by measuring diastolic blood pressure (DBP), renovascular responses by measuring p-aminohippurate (PAH) clearance and adrenal responses by measuring plasma aldosterone. After receiving captopril for 66 h the hypertensive subjects showed a significantly (P less than 0.004) enhanced blood pressure response to the infused ANG II but not to noradrenaline when compared with the response before captopril. ANG II (3 pmol kg-1 min-1) also produced a significantly (P less than 0.03) greater reduction in PAH clearance after (-194 +/- 40 ml/min) compared with before (-104 +/- 15 ml/min) captopril. These results suggest that the responsiveness to ANG II in these two target tissues is determined by the circulating ANG II level. In the adrenal gland the aldosterone responses to ANG II also were significantly greater after (P less than 0.01) than before captopril (increment at 3 pmol kg-1 min-1: 660 +/- 88 vs 381 +/- 94 pmol/l). These results are in distinct contrast with the responses previously reported for normotensive subjects and support the hypothesis that the regulation of aldosterone secretion is altered in subjects with essential hypertension.
Adrenal Glands↗
Abnormal regulation of parathyroid cell secretion and proliferation in primary cultures of bovine parathyroid cells.
We developed a method for establishing primary monolayer cultures of bovine parathyroid cells, characterized the cells morphologically, and studied the effects of calcium on PTH secretion and cellular proliferation. Fresh bovine parathyroid glands were enzymatically and mechanically dispersed, as previously described, using sterile solutions and apparatus. The cells were then plated at a density of 2.5 X 10(5) cells/cm2 into cluster wells and incubated at 37 C in a medium containing Dulbecco's Modified Eagle's and Ham's F-12 medium, 15% newborn calf serum, Hepes, antibiotics, and insulin. In 3-4 days, a near-confluent monolayer of polygonal-shaped cells with less than 10% fibroblasts was achieved. Electron microscopy revealed a homogeneous cell population containing electron-dense secretory granules as well as abundant rough endoplasmic reticulum and mitochondria, with no evidence of organelle swelling. Two parameters of cellular proliferation increased significantly in culture; viable cell number increased from 289,000 +/- 63,000 to 530,000 +/- 60,000 per well (P less than 0.02), and cellular protein increased from 40.00 +/- 1.40 to 177.60 +/- 57.10 micrograms/well (P less than 0.035). On an hourly basis, cultured cells showed a greater secretory rate than that of acutely dispersed cells (9.3 vs. 5.0 ng/10(5) cells X h, respectively). Unlike normal bovine parathyroid cells, however, high calcium concentrations inhibited PTH secretion only slightly (21.2 +/- 4.7%) and had no effect on cellular proliferation. Addition of the divalent cation ionophore A23187, on the other hand, inhibited PTH release by 50% or more, suggesting that the secretory apparatus is responsive to increases in the cytosolic calcium concentration. The present study reveals that bovine parathyroid cells in primary culture proliferate and secrete PTH in vitro. These cells display, however, a generalized decrease in sensitivity to the suppressive effects of extracellular calcium on hormonal secretion and cellular proliferation comparable to that of pathological parathyroid tissue. Thus, this cell culture system may provide a useful model for investigating the relationship between secretion and proliferation in normal and abnormal tissue.
Animals↗
Bioavailability of thyroid hormones from oral replacement preparations.
We evaluated gastrointestinal absorption in normal subjects of T4 and T3 from synthetic T3 tablets (Cytomel, SKF), desiccated thyroid tablets (Armour), thyroglobulin tablets (Proloid, Warner-Chilcott) and synthetic L-T4 tablets (Synthroid, Flint and Levothroid, Armour). Measurements of serum T4 and T3 concentrations and free hormone indices were made at multiple times after tablet ingestion, and T3 content in tablets was measured by radioimmunoassay. The time to peak serum T3, and the 26 hr intergrated increment in serum T3, Corrected for the amount if T3 ingested, were not significantly different for 75 micrograms of synthetic T3, 6 grains of desiccated thyroid (containing 99 micrograms T3) and 5 grains of thyroglobulin (containing 90 micrograms T3), the mean integrated increment values for the biological preparations being within 12% of those for synthetic T3. The peak serum T4 concentration, the time to peak T4, and 48 hr integrated increments in serum T4 and T3 were similar after 3 mg of Synthroid and Levothroid. The mean peak serum Free T3 Index after 75 micrograms T3, 500, was much higher than the mean peak Free T3 Index after 3 mg T4, 290. The time to peak Free T3 Index was much less after 75 micrograms T3, 2 hr, than the time to peak after 3 mg T4, 2 days. These results indicate that the time course and extent of T3 absorption do not differ, whether the T3 is given as the synthetic iodothyronine or as part of the thyroid protein, thyroglobulin. This approach appears to be useful in determining bioavailability of thyroid hormones from oral preparations and to assess the possibility of thyroid hormone malabsorption.
Biological Availability↗
Cyclosporine A and prednisone-associated osteoporosis in heart transplant recipients.
Immunosuppressive therapy with cyclosporine A or prednisone produces bone loss in some animal models. Although we have clinically observed osteoporotic fractures in our heart recipients, the effects of cyclosporine and prednisone on bone density in transplant populations has not been fully elucidated. This study was undertaken to examine indexes of mineral metabolism and bone mineral density (BMD) in heart transplant recipients referred for evaluation of possible bone disease. Twenty of 93 patients who underwent heart transplantation at our institution were evaluated for osteoporosis. Sixteen of these patients (eight men; eight women) were included in this cross-sectional study (two patients were excluded because of hyperparathyroidism, and two patients were excluded because severe fractures prevented BMD from being measured). The mean age of the heart transplant recipients was 52.4 +/- 2.2 years, and the study was conducted a mean of 33.4 +/- 4.6 (men) and 19.0 +/- 7.0 (women) months after heart transplantation. Forty-four percent of these heart transplant recipients were seen clinically with fractures. Biochemical tests of skeletal homeostasis and BMD measurements with dual energy x-ray absorptiometry were performed. In male and female patients, the indexes of mineral metabolism showed (mean +/- sem) osteocalcin levels of 9.60 +/- 2.3 micrograms/L and 9.46 +/- 1.9 micrograms/L (normal: men, 6.39 +/- 0.69 micrograms/L; women, 5.87 +/- 0.71 micrograms/L) and intact parathyroid hormone levels of 48.8 +/- 10.3 ng/L and 63.4 +/- 10.7 ng/L (normal: men, 26.8 +/- 3.3 ng/L; women, 30.7 +/- 2.1 ng/L), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Bone Density↗
Modeling fracture risk using bone density, age, and years since menopause.
INTRODUCTION: Preventive strategies are essential for reducing the incidence of osteoporosis and its consequences. However, simple algorithms that predict an individual's future risk of fractures are scarce. The purpose of the study was to define a clinical decision aid that enables physicians to project an individual's life-time fracture risk and therefore institute preventive therapies. METHODS: A predictor equation for bone loss was developed using bone mineral density (BMD), age, years since menopause, and weight. This was applied to normal and osteoporotic women, ages 40-80 years (n = 117) screened for osteoporosis studies. RESULTS: A spinal BMD cutoff of 0.86 gm/cm2 had a sensitivity of 90% and a specificity of 60% for detecting subjects with vertebral fractures and was therefore defined as a high-risk BMD. Using the parameter estimates from the above equation and an individual's clinical data, we derived prediction curves to forecast the age at which that individual would reach the above defined high-risk BMD, and therefore that person's expected number of remaining life-years at high risk for fractures. CONCLUSIONS: This study proposes a conceptual framework for the development of a clinical decision aid to provide guidelines for the prevention of osteoporosis. A longitudinal study that incorporates other variables such as prevalent fractures and biochemical markers of bone turnover would further validate this model and enhance its application.
Adult↗