Normal Human Parathyroid Research Articles

Regulation of parathyroid hormone release in normal and pathological parathyroid cells exposed to modulators of protein kinase C.

Effects of the protein kinase C activating phorbol ester 12-O-tetradecanoyl phorbol 13-acetate and the inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7) on parathyroid hormone (PTH) release were studied in normal bovine and pathological human parathyroid cells. An increase of extracellular Ca2+ from 0.5 to 3.0 mmol/l inhibited PTH release by 60% in the bovine cells with half maximal effect (ED50) at 1.31 mmol/l. This inhibition reached less than 50% in the cells from patients with primary and uremic hyperparathyroidism, and the ED50 values were 1.49 and 1.42 mmol/l, respectively. The phorbol ester (0.1 mumol/l) made secretion insensitive to changes of extracellular Ca2+, an action counteracted by H-7 (50 mumol/l) in the bovine cells, whereas H-7 alone had no effects. The phorbol ester and H-7 had opposite actions on regulation of PTH release also from cells from patients with hyperparathyroidism. However, in pathological cells H-7 alone improved Ca2+ inhibition of secretion by stimulating release in low Ca2+ concentrations and decreasing the ED50 values. The magnitude of changes in ED50 values by H-7 increased with the severity of the secretory disturbance of the pathological cells. The results indicate that increased protein kinase C activity may be a factor of importance in the pathophysiology of hyperparathyroidism.

Neomycin interacts with Ca 2+ sensing of normal and adenomatous parathyroid cells

Effects of the polyvalent cationic antibiotic neomycin on regulation of the cytoplasmic Ca 2+ concentration ([Ca 2+] i) were studied in normal and adenomatous human, and bovine parathyroid cells. Parathyroid hormone (PTH) release was also measured in the bovine cells. Elevation of extracellular Ca 2+ from 0.5 to 3 mM caused biphasic increase of [Ca 2+] i and inhibition of PTH release. In low external Ca 2+ neomycin inhibited PTH release and virtually only triggered the [Ca 2+] i transient. In contrast [Ca 2+] i was lowered and PTH release stimulated by neomycin in the presence of 3.0 mM Ca 2+ or 7 mM Mg 2+. These actions of Ca 2+ and neomycin on [Ca 2+] i were qualitatively similar but less pronounced in the adenomatous than normal human parathyroid cells. Some effects of neomycin were thus similar to those induced by other cationic agents interacting with the Ca 2+ receptor mechanism on the parathyroid cell surface, whereas others may involve phospholipase C inhibition, protein kinase C activation or a direct reduction of the Ca 2+ influx.

Interaction of monoclonal antiparathyroid antibody with Ca 2+ agonistic actions of Mn 2+ in normal human parathyroid cells

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn 2+ Interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca 2+, 3mM Mn 2+ induced a rapid transient increase in cytoplasmic Ca 2+ [Ca 2+ i] followed by quenching of the fluorescence from the Ca 2+ indicator fura-2 as Mn 2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca 2+ i transient and considerably delayed the entry of Mn 2+. The results support the presence of a cation-sensltive receptor mechanism on parathyroid cells and indicate that the antibody C11 not only blocks the interaction between Ca 2+ and this receptor mechanism but also that of Mn 2+.

Monoclonal antiparathyroid antibodies revealing defect expression of a calcium receptor mechanism in hyperparathyroidism.

AbstractMonoclonal antibodies were generated with a hybridoma technique after immunization of mice with intact human parathyroid cells. Three antibodies of the IgG type reacted in immunohistochemistry and immunofluorescense with parathyroid epithelial cells and proximal tubule cells of the kidney but not with a large number of other human tissues. Adenomatous and hyperplastic parathyroid glands of patients with hyperparathyroidism (HPT) demonstrated a reduced immunohistochemical reactivity with the antibodies as compared to the intense staining of normal human parathyroid tissue. Experiments with dispersed parathyroid cells from the normal and pathological glands showed that 2 of the antibodies blocked the effects of ambient calcium on cytoplasmic calcium and parathyroid hormone release. This indicates that the antibodies interfere with a newly recognized receptor mechanism of parathyroid cells involved in the sensing and gating of calcium and thereby also in the regulation of parathyroid hormone release. A reduced expression of the putative calcium receptor mechanism of abnormal parathyroid cells may, thus, be principally responsible for the hypercalcemia of HPT by causing a decreased sensitivity to extracellular calcium of the parathyroid hormone release from pathological parathyroid glands. The antibodies should improve the parathyroid histological diagnosis by their ability to identify and to distinguish between normal and abnormal parathyroid tissue.

Hyperparathyroidism is associated with reduced expression of a parathyroid calcium receptor mechanism defined by monoclonal antiparathyroid antibodies.

Parathyroid tissue from patients with hyperparathyroidism (HPT) exhibited reduced immunohistochemical reactivity with monoclonal antiparathyroid antibodies, previously shown to stain intensely the surface of normal human parathyroid cells and to interfere with a receptor mechanism of these cells which is involved in the sensing and gating of Ca2+. Parathyroid hormone (PTH) release and cytoplasmic Ca2+ concentrations (Ca2+i) of dispersed cells from the pathological parathyroid glands had right-shifted dependencies on extracellular Ca2+, and exposure to the antibodies rendered both Ca2+i and PTH release almost completely insensitive to changes in ambient Ca2+. The results suggest that reduced expression of a parathyroid calcium receptor mechanism may be an important cause for the aberrant PTH release in HPT.

Cytoplasmic Ca2+ concentration of single normal human and bovine parathyroid cells measured by dual wavelength microfluorometry.

Dual wavelength microfluorometry was utilized to measure the cytoplasmic calcium concentration (Cai2+) of single parathyroid cells loaded with the indicator fura-2. The method enabled the first registrations of Cai2+ of normal human parathyroid cells, available only in minute numbers. At 0.5 mM extracellular Ca2+, the Cai2+ levels were similar in normal human and bovine cells. Both cell types responded with an initial Cai2+ transient followed by a sustained increase when raising extracellular Ca2+ to 3.0 mM. The sustained effect exhibited a sigmoidal relation to extracellular Ca2+ in the 0.5-3.0 mM range. Although the increase was somewhat greater in the human cells, the half maximal responses were obtained at almost identical extracellular Ca2+ concentrations. Whereas K+ depolarization decreased Cai2+, the Ca2+ channel blocker D-600 had dual actions, raising Cai2+ at 0.5 mM Ca2+ and decreasing it at 3.0 mM Ca2+, and the effects were similar in the bovine and human cells. The present experimental approach verified the validity of utilizing bovine cells as controls in studies of human parathyroid tissue and it appears suitable for analysis of the role of different subpopulations of parathyroid cells in the abnormal parathyroid tissue of patients with hyperparathyroidism.

Dimethyl sulfoxide increases cytoplasmic Ca 2+ concentration and inhibits parathyroid hormone release in normal bovine and pathological human parathyroid cells

The acute effects of dimethyl sulfoxide (DMSO) on parathyroid hormone (PTH) release and the cytoplasmic Ca 2+ concentration (Ca 2+ i) were studied in dispersed bovine cells and cells isolated from human parathyroid adenomas. At extracellular Ca 2+ concentrations in the 0.5–3.0 mM range, but not at less than 25 nM, addition of 2% DMSO caused a rapid rise of Ca 2+ i. This effect corresponded to an inhibition of PTH release and there was a strong negative correlation between Ca 2+ i and secretion. The actions of DMSO on Ca 2+ i and PTH release were less pronounced in the pathological human cells. The data are consistent with a DMSO effect on the Ca 2+-sensor function of the parathyroid cell, possibly mediated by an altered plasma membrane fluidity.

Acute parathyroid hormone secretory dynamics: hormone secretion from normal primate and adenomatous human tissue in response to changes in extracellular calcium concentration.

PTH secretion has been evaluated extensively using short term incubation techniques, but these methods cannot be used to adequately evaluate the early phases of PTH secretion. We developed a dispersed cell perifusion system to study these acute secretory events. Responses to low calcium conditions were studied using dispersed cells from normal primate and adenomatous human parathyroid tissue. When these cell were perifused with 1.0 mM calcium medium, PTH secretion was stable, but increased within minutes in a dose-dependent manner in response to a lowering of extracellular calcium concentrations. Similarly, PTH secretion quickly declined when cells were exposed to higher extracellular calcium concentrations. In studies of cells from 11 human adenomas, the mean maximum secretion in response to 0.25 mM calcium conditions was 587 +/- 330% (+/- SD) of that in response to 1.0 mM calcium. The magnitude of the response to low calcium stimulation was variable, and cells from two additional adenomas failed to respond to low calcium stimulation despite responses to other secretogogues. Variation in the rate of increase in stimulated PTH secretion was also found among the adenomas examined (128 +/- 95% of baseline/min), and the rate of increase in PTH release and the eventual maximum rate of release were positively correlated. In studies of cells from 4 normal primates (rhesus macaque), the maximum response to 0.25 mM Ca2+ (544 +/- 118% of baseline) and the rate of increase in PTH secretion (95 +/- 35% of baseline/min) were similar to those in adenomatous human tissue. These results indicate that 1) acute PTH secretion can be studied in vitro, and that it appears to be similar to the in vivo process; 2) there is a wide variation among adenomatous tissues in the magnitude of secretion stimulated by low calcium concentrations; 3) in addition to varied magnitude of secretion, the rate at which hormone secretion increases in response to a low calcium stimulus varied among adenomas; and 4) PTH secretion from human adenomatous tissue is similar to that from normal primate tissue in both the rate and magnitude of response.

Paradoxical effects of K + and D-600 on parathyroid hormone secretion and cytoplasmic Ca 2+ in normal bovine and pathological human parathyroid cells

The effects of K + and the Ca 2+ channel blocker D-600 on parathyroid hormone (PTH) release and cytoplasmic Ca 2+ activity (Ca 2+ i) were measured at different Ca 2+ concentrations in dispersed parathyroid cells from normal cattle and from patients with hyperparathyroidism. When the extracellular Ca 2+ concentration was raised within the 0.5–3.0 mM range Ca 2+ i increased and PTH secretion was inhibited. There was also a stimulatory effect of Ca 2+ on secretion as indicated by a parallel decrease of Ca 2+ i and PTH release when extracellular Ca 2+ was reduced to less than 25 nM. Addition of 30–50 mM K + stimulated PTH release and lowered Ca 2+ i. The effect of K + was less pronounced in the human cells with a decreased suppressability of PTH release. The Ca 2+ channel blocker D-600 had no effect on Ca 2+ i and PTH release in the absence of extracellular Ca 2+. However, at 0.5–1.0 mM Ca 2+, D-600 increased Ca 2+ i and inhibited PTH release, whereas the opposite effects were obtained at 3.0 mM Ca 2+. The transition from inhibition to stimulation occurred at a higher Ca 2+ concentration in the human cells and the right-shift in the dose-effect relationship for Ca 2+-inhibited PTH release tended to be normalized by D-600. It is suggested that K + stimulates PTH release by increasing the intracellular sequestration of Ca 2+ and that the reduced response in the parathyroid human cells is due to the fact that Ca 2+ i already is lowered. D-600 appears to have both Ca 2+ agonistic and antagonistic actions in facilitating and inhibiting Ca 2+ influx into the parathyroid cells at low and high concentrations of extracellular Ca 2+, respectively. D-600 and related drugs are considered potentially important for the treatment of hyperparathyroidism.

Calcium messenger system: an integrated view.

Calcium messenger system: an integrated view.

Pancreatic polypeptide (PP) immunoreactivity in human parathyroid culture media

Media from cultures of normal and abnormal human parathyroid fragments were assayed for parathyrin (PTH) and pancreatic polypeptide (PP) using sensitive radioimmunoassays. PP immunoreactivity was present in media (Day 6–7 in vitro) from cultures of 3 10 adenomas and 6 6 3° hyperplastic glands (mean = 126. fmole/mg protein/day) (range = 6.–675.), and was not suppressed by 0 — 3 m M calcium challenge. PP was undetectable in media from cultures of one parathyroid carcinoma, one 1° hyperplasia, and one normal parathyroid. Medium C-terminal PTH levels were quite variable (26.–2,545,000. pg/ mg protein/day). Presence of PP immunoreactivity in media from cultures of some hyperplastic parathyroids and some parathyroid adenomas suggests that PP may be released from these tissues in vitro. The significance of elevated PP levels in the MEA syndromes may be of special clinical relevance to this observation.

Autografts of normal and hyperplastic human parathyroid

Autografts of normal and hyperplastic human parathyroid

Effect of calcium on cyclic nucleotide phosphodiesterase in parathyroid tissue

The hydrolysis of cyclic AMP and cyclic GMP by homogenates of normal bovine parathyroid gland and human parathyroid adenomas was decreased by EGTA. When supernatants were chromatographed on DEAE-cellulose it was found that sheep brain calmodulin in the presence of calcium stimulated cyclic AMP and cyclic GMP phosphodiesterase activity. The response to calmodulin in two human parathyroid adenomas was less than that in normal bovine parathyroid. Calmodulin was detected in heat-treated supernatants of 11 parathyroid adenomas by its ability to activate calmodulin-free sheep brain phosphodiesterase. The results suggest a role for calcium in the hydrolysis of cyclic nucleotides in parathyroid tissue.

Autografts of normal and hyperplastic human parathyroid: experience in eleven patients with immunoassay monitoring of function in seven.

Parathyroid autografts were implanted in 11 patients at this institution. Four patients had total or subtotal parathyroidectomy and implantation for parathyroid hyperplasia or adenoma, while 7 had one or more parathyroids removed and implanted during total thyroidectomy for carcinoma of the thyroid. Hyperplastic parathyroid was implanted in 4 instances and normal parathyroid in 7. Recurrent hyperparathyroidism occurred in 1 patient with an implanted adenoma who was returned to a normocalcemic state by partial reexcision ("titration") of the implanted tissue. All implants were placed in muscle pockets, a single site being used for patients receiving normal parathyroid tissue, and multiple sites for patients receiving hyperplastic glands. Those patients receiving forearm implants were followed up by serial parathyroid hormone determinations in venous blood drawn from both arms. In all cases, elevated levels were found on the implanted side, as compared to the contralateral control side.

Influence of Vitamin D3, 1,25-Dihydroxyvitamin D3, and 24,25-Dihydroxyvitamin D3on Parathyroid Hormone Secretion, Adenosine 3 ′,5′-Monophosphate Release, and Ultrastructure of Parathyroid Glands in Organ Culture*

The influence of supraphysiological and physiological concentrations of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on the endocrine activity of normal porcine parathyroid glands and human parathyroid adenomas in organ culture was studied. In the medium, parathyroid hormone (PTH) and cAMP were measured radioimmunologically and correlated with ultrastructural changes of the tissue. During incubations with 1,25(OH)2D3 (range, 0.03–40.0 ng/ml), PTH secretion decreased uniformly from 100% to an average of 60%. In a dose-response experiment, 0.015 ng/ml 1,25(OH)2D3 was the lowest concentration causing a significant inhibition. After elimination of this steroid, PTH secretion increased to nearly normal values again within 2 h. cAMP release showed parallel changes. Vitamin D3 (4000 ng/ml) and 24,25(OH)2D3 (40.0 and 5.0 ng/58:44ml) had no effect. De novo synthesis of the secreted PTH was checked by incubation with [75Se]methionine; its biological potency was es...

Acid Phosphatase Activity in Mammalian Parathyroid Glands

It has been suggested that the parathyroid secretory granules might represent lysosomes. Since lysosomes are known to contain acid phosphatases (AcPase), this enzymatic activity was investigated with a modified Gomori lead method with β-glycerophosphate as the substrate to determine the relationship of secretory granules to lysosomes in adenomatous and hyperplastic (primary chief cell type) human, and normal bovine, human, and rat parathyroid glands.Intracellular activity was demonstrated in the lipofuchsin granules and in rare, membrane-limited bodies consistent with lysosomes (Figs. 1 and 2). These lysosomal granules were often irregular in shape and 450 to 850 mμ in diameter. Lead precipitate was also observed in association with some Golgi membranes and cisternae and in a few small Golgi vesicles (Fig.3). There was strong AcPase reactivity in the lumenal cell membranes of the capillary endothelium (Fig. 4) though the cell membranes adjacent to the basement membranes and those of the parathyroid parenchymal cells were devoid of enzymatic activity. No activity was present in the numerous small secretory granules or in the granular endoplasmic reticulum (Figs. 1 and 2).