Inhibition Of Parathyroid Hormone Release Research Articles

Hypocalcemia in Infants and Children

Hypocalcemia in Infants and Children

Effects of calcium channel modulators on the regulation of cytoplasmic Ca2+ and hormone secretion of parathyroid cells.

Effects of Ca(2+)-channel modulators were examined in human, bovine and murine parathyroid tissue. In 0.5 mM external Ca2+, 100 microM verapamil inhibited parathyroid hormone release, stimulated uptake and efflux of 45Ca and raised cytoplasmic Ca2+ concentration ([Ca2+]i). However, in 3.0 mM Ca2+ the drug only affected efflux. Methoxyverapamil (50 microM) inhibited parathyroid hormone release in 0.5 mM but stimulated secretion in 3.0 mM Ca2+. BAY K 8644 (10 microM) had similar actions as verapamil on parathyroid hormone release and [Ca2+]i, whereas nifedipine (1 microM) and diltiazem (50-100 microM) lacked effects. Despite the lack of voltage-dependent Ca2+ channels in parathyroid cells, drugs with established actions on such channels affect [Ca2+]i and parathyroid hormone release. However, these actions are not sufficiently pronounced and tissue specific to allow their use for correcting hyperparathyroidism.

Fluoride interactions with stimulus-secretion coupling of normal and pathological parathyroid cells.

Effects of the GTP binding protein (G-protein) activator NaF on parathyroid hormone (PTH) release, cytoplasmic Ca2+ concentration ([Ca2+]i) and cAMP content of bovine as well as normal and pathological human parathyroid cells were studied using precautions to avoid CaF2 precipitation. In 0.5 mM external Ca2+, NaF inhibited PTH release and lowered the cAMP content by 50-70% of the effects attained with 3.0 mM Ca2+. The NaF-induced increase of [Ca2+]i was considerably smaller than that obtained with rise of external Ca2+. It seems likely that NaF activates the inhibitory G1-protein involved in the regulation of cAMP generation. However, it is unclear whether the sluggish rise of [Ca2+]i induced by NaF is due to a direct effect of a G-protein on Ca2+ entry, or somehow related to the G-protein mediated formation of inositol 1,4,5-trisphosphate, which is part of the signal transduction pathway normally initiated by Ca2+ binding to its receptor on the parathyroid cell surface. Inhibition of PTH release by NaF probably results from the combined effects on [Ca2+]i and cAMP content. In hyperparathyroidism (HPT) the actions of NaF were not markedly affected despite severe impairments of Ca(2+)-inhibited PTH release and Ca2+ triggered increase of [Ca2+]i. Consistent with observations of down regulation of the parathyroid Ca2+ receptor in HPT, the present results indicate that the disease perturbs signal transduction at a level proximal to the site of action for NaF.

Ga 3+ inhibits parathyroid hormone release without interacting with the Ca 2+ receptor of the parathyroid cell

Gallium nitrate is an antihypercalcemic agent with established actions on bone. The effects of Ga(NO3)3 on parathyroid hormone (PTH) release, cytoplasmic Ca2+ concentration ([Ca2+]i) and cAMP production of enzymatically dispersed parathyroid cells from bovine as well as normal and pathological human parathyroid glands have now been studied. Ga3+ at 200 microM inhibited PTH release whereas 600 microM NO3- had no effect. The inhibition was additive to that obtained by elevating extracellular Ca2+. Unlike Ca2+, Ga3+ failed to increase [Ca2+]i or reduce cAMP formation. The results indicate that Ga3+ inhibits PTH release by a mechanism other than activation of the cation receptor of the parathyroid cells. This mechanism may contribute also to inhibition by other cations.

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.

Cellular physiology and pathophysiology of the parathyroid glands

This report provides insight into parathyroid gland physiology and the pathophysiology of hyperparathyroidism (HPT). Increases in the extracellular calcium concentration constitute the primary physiological signal for inhibition of parathyroid hormone (PTH) release. Transduction of the external signal into a cellular response involves activation of a cation receptor mechanism on the plasma membrane with rapid rise in the cytoplasmic calcium concentration of the cells. This recently discovered parathyroid calcium receptor has been characterized as a glycoprotein of unusually high molecular weight, which may play a key role in calcium homeostasis since it is also expressed in the kidney and placenta. Binding of external calcium to the receptor is associated with mobilization of intracellular calcium as well as calcium influx into the cells and phosphoinositol hydrolysis. These events rapidly interfere with the release process through essentially unknown mechanisms and probably also at sustained stimulation inhibit PTH gene transcription. The relative calcium insensitivity of the PTH release in HPT is associated with a deranged regulation of cytoplasmic calcium within pathological parathyroid cells. The molecular basis for this disturbance comprises down regulation of the cation receptor, whereby external calcium is translated into abnormally low levels of cytoplasmic calcium and insufficient inhibition of PTH release. Studies on expression of the functionally important cation sensing glycoprotein and its associated cellular signal systems may provide novel means for interference with the pathophysiological derangements of HPT.

Inhibition of Parathyroid Hormone Release by Maitotoxin, a Calcium Channel Activator

Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.

Calcium-agonistic action of Mn 2+ in the parathyroid cell

The effects of 3.0 mM Mn 2+ on parathyroid hormone (PTH) release, 45Ca fluxes and the cytoplasmic Ca 2+ concentration (Ca 2- i) were studied in vitro with use of parathyroid glands from rats and cattle. Mn 2+ inhibited PTH release by about 70 and 60% at Ca 2+ concentrations of < 10 nM or 0.5 mM, respectively, and like the inhibitory effect of Ca2+, that of Mn2+ was rapid and reversible. Mn 2+-induced inhibition of the PTH release was associated with reduction of 45Ca uptake by about 50% and a marked but temporary increase in 45Ca efflux. Mn 2+ induced a rapid transient increase in Ca 2+ i provided that the intracellular release of Ca 2+ had not already been triggered by previous exposure to 3.0 mM Ca 2+. This initial action was apparently followed by influx of Mn 2+, since pronounced quenching of the fluorescence from the Ca 2+ indicator fura-2 was observed. The results indicate that Mn 2+ behaves in several respects like a calcium analogue in parathyroid cells and may inhibit PTH release by interfering directly with the secretory machinery.

Antibodies to an alpha subunit of skeletal muscle calcium channels regulate parathyroid cell secretion.

We have shown previously that Ca2+-channel agonists, which open Ca2+ channels, inhibit parathyroid hormone (PTH) secretion from dispersed bovine parathyroid cells, whereas Ca2+-channel antagonists, which close Ca2+ channels, stimulate PTH release. We now have tested the effects of mouse antibodies specific for purified alpha subunits of rat skeletal muscle Ca2+-channel proteins on PTH secretion by bovine parathyroid cells in vitro. Mouse antisera (MC-2, MC-3, MC-4) blocked the secretion of PTH from parathyroid cells incubated with 0.5 mM Ca2+ ions. Affinity-purified MC-4 antibodies inhibited PTH release in a concentration-dependent manner. Incubation of parathyroid cells with pertussis toxin markedly reduced MC-4-dependent inhibition of PTH secretion. Parathyroid cell membrane proteins were fractionated by NaDodSO4/polyacrylamide gel electrophoresis under either reducing or nonreducing conditions and immunoblotted with MC-4 antiserum. Antibodies bound to one major band of protein with Mr approximately equal to 150,000. These results suggest that the antibodies bind to Ca2+-channel alpha subunits and act as agonists that open the channels and inhibit PTH release.

Functional dedifferentiation of parathyroid cells results both from a defective regulation and action of cytoplasmic Ca2+.

Monolayer culture of bovine parathyroid cells for 24 hours resulted in a right-shift of the dose-effect relationships for Ca2+-inhibition of parathyroid hormone (PTH) release and the dependence of the cytoplasmic Ca2+ concentration (Cai2+) on extracellular Ca2+ as well as in a less suppressible hormone release. After 4 days of culture, hormone secretion was almost non-suppressible and Cai2+ increased poorly in response to a rise in extracellular Ca2+. Ionomycin, a Ca2+ ionophore, raised Cai2+, but there was only a small inhibition of PTH release and the correlation between Cai2+ and secretion was weak. A deteriorated Cai2+ regulation and a decreased inhibitory action of cytoplasmic Ca2+ on PTH release were also found in cells from human parathyroid adenomas. Functional dedifferentiation of the parathyroid cell thus results from both defective regulation and action of cytoplasmic Ca2+.

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.

Effect of the phorbol ester TPA on PTH secretion Evidence for a role for protein kinase C in the control of PTH release

Parathyroid hormone (PTH) secretion is stimulated by low extracellular calcium (Ca 2+) in association with a reduction in cyosolic Ca 2+, indicating that this cell type does not conform to classical models of stimulus-secretion coupling. We used the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate), which directly activates protein kinase C, to investigate the possible role of this enzyme in the unusual secretory properties of the parathyroid cell. TPA causes a dose-dependent stimulation of PTH release inhibited by high extracellular Ca 2+ (EC 50 = 10 nM) but has relatively little effect on secretion stimulated by low Ca 2+. This effect was mimicked by the β4-isomer of phorbol 12,13-didecanoate which also activates kinase C, but not by the α4-isomer, which has no effect on this enzyme. TPA does not modify cellular cAMP or cytosolic Ca 2+ in the parathyroid cell indicating that its effects on PTH secretion are not mediated indirectly via changes in these second messengers. These results suggest that inhibition of PTH release at high Ca 2+ might be related to a reduction in protein kinase C activity which can be overcome when the enzyme is directly activated by TPA.

Inhibition of parathyroid hormone release from dispersed bovine cells by ouabain

Ouabain, at concentrations of 3 × 10 −4–10 −3 M, inhibited secretion of PTH ∼50% in a freshly dispersed bovine parathyroid cell preparation. This inhibition was found with both unstimulated and secretagogue-stimulated PTH release. Reductions in PTH secretion were found at all concentrations of Ca ++ tested between 0.3 mM and 2.0 mM and in the presence of the divalent cation chelators EDTA and EGTA, indicating that extracellular Ca ++ is not an absolute requirement for the inhibition. The ouabain inhibition did not appear to be mediated through changes in either adenylate cyclase activity or total cellular cAMP, implying a locus distal to the generation of this cyclic nucleotide. The data suggest that transmembrane potential and/or distribution of monovalent cations across the plasma membrane is important in the maintenance of PTH secretion. The mechanisms involved in this control do not appear to involve extracellular Ca ++ directly.

Sodium nitroprusside inhibition of parathyroid hormone release is not mediated through cyclic GMP

Sodium nitroprusside effected a rapid, dose-dependent increase in intracellular cGMP accumulation in freshly dispersed bovine parathyroid cells. The effect was half-maximal between 10 −4 and 3 × 10 −4M, maximal at 3 × 10 −3M nitroprusside and could be amplified (∼50%) by the addition of methylisobutylxanthine (4 × 10 −4M). The dose-response characteristics were similar to those previously described for the inhibition of cAMP accumulation and PTH release by this agent. Neither dibutyryl cGMP (10 −3M) nor 8′-bromo-cGMP (10 −3M) mimicked the inhibitory effect of nitroprusside on cAMP accumulation or PTH release. Dose-dependent stimulation of guanylate cyclase was found in a particulate preparation of parathyroid cells; activity was maximal at 10 −4M nitroprusside while higher concentrations appeared to inhibit the enzyme. Nitroprusside significantly reduced both (−)isoproterenol and guanine nucleotide-stimulated adenylate cyclase activity in the particulate preparation with maximum inhibition between 10 −3–10 −2M. cGMP concentrations as high as 10 −4M did not affect agonist-stimulated cAMP synthesis. Thus, although the kinetic and dose-response characteristics of the nitroprusside effect on cGMP suggest a linkage to its previously described effects on cAMP and PTH secretion, no direct evidence was found to indicate a causal relationship between the two. Rather it would appear that the effects on the adenylate and guanylate cyclase enzymes occur in parallel, possibly the result of some common primary perturbation of cellular physiology.

Stimulation of in vitro release of calcitonin from rat and human C-cells by cimetidine.

Previous reports have suggested that the H2-receptor blocker, cimetidine, can inhibit parathyroid hormone (PTH) release. The present studies were designed in an attempt to see whether cimetidine affects secretion of calcitonin (CT) as well. Entire thyroparathyroid glands from 8-day-old baby rats or small pieces of a human medullary thyroid carcinoma were incubated at 37 C for up to 8 h in chemically defined culture medium gassed with 95% 02-5% CO2. With rat thyroparathyroids, both CT and PTH released into medium were measured using RIAs that detect that rat hormones. CT secreted from human C-cells was measured using an RIA for human CT. The results were: (A) As we had found previously, cimetidine at doses of 3 x 10(-3) M and 9 x 10(-3) M inhibited PTH release from rat thyroparathyroids by as much as 50-60% (p less than 0.05 - less than 0.001). In these same experiments, cimetidine produced increases of approximately 1- to 5-fold in the CT levels found in the medium (p less than 0.05 - less than 0.001). (B) Fragments of human medullary thyroid carcinoma incubated for 3 h in the presence of 10(-5) M cimetidine released considerably more CT than tissue incubated in control medium (48 +/- 9.3 vs 21 +/- 3.6 ng CT/microgram tissue protein, p less than 0.05). Our results show that cimetidine in vitro not only can inhibit secretion of PTH but also apparently can promote the release of CT from both rat and human C-cells. Whether this effect is mediated by H2 receptors and whether it is of any physiological significance awaits clarification.

Effects of vitamin D metabolites on bovine parathyroid hormone release in vitro.

We evaluated the effects of 1 alpha,25-dihydroxycholecalciferol (1,25(OH)2D3), 24R,25-dihydroxycholecalciferol (24,25(OH)2D3), and 25-hydroxycholecalciferol (25(OH)D3) on the release of parathyroid hormone (PTH). Bovine parathyroid tissues were incubated in vitro for 4 h in low-calcium (1.0 mM) medium. 1,25(OH)2D3 ((10(-9)-10(-12)M), 24,25(OH)2D3 (10(-6)-10(-8)M), and 25(OH)D3 (5 X 10(-7)-5 X 10(-9)M) inhibited PTH release. Inhibition by all metabolites was concentration and time dependent. On a molar basis, 1,25(OH)2D3 was the most potent metabolite, being at least 100 times more potent than 24,25(OH)2D3 and 25(OH)D3; 24,25(OH)2D3 was about 5 times more potent than 25(OH)D3 at concentrations producing 65% inhibition. Inhibition by high concentrations of metabolites was evident by 1 h of incubation; inhibition was progressive throughout incubation, and maximal suppression to 30-40% of control occurred during the fourth and final hour of incubation. 1,25(OH)2D3 (10(-11) M), a low concentration that did not inhibit secretion, transiently stimulated release. In conclusion, under conditions of low-calcium-stimulated PTH release, 1,25(OH)2D3, 24,25(OH)2D3, and 25(OH)D3 inhibited PTH release, 1,25(OH)2D3 was the most potent inhibitor.

Diabetic ketoacidosis: Induction of hypocalcemia and hypomagnesemia by phosphate therapy

A nine year old boy with previously undiagnosed diabetes mellitus presented with severe ketoacidosis. His hyperglycemia (plasma glucose = 786 mg/dl), acidosis (arterial pH = 6.86), dehydration and coma responded well to therapy with intravenous fluids, bicarbonate and insulin. Potassium supplementation was given as a phosphate salt. Despite marked clinical and biochemical improvement, 28 hours after therapy was initiated he was found to have profound hypocalcemia (2.6 meq/liter), hypomagnesemia (0.8 meq/liter) and hyperphosphatemia (9.2 mg/dl). All three electrolyte levels had been normal upon admission, and they were gradually corrected with appropriate supplementation of calcium and magnesium and discontinuation of the intravenous phosphate. We interpret these iatrogenic electrolyte abnormalities in the patient described to have been the result of the massive phosphate load administered, resulting not only in hypocalcemia, but also in hypomagnesemia that inhibited parathyroid hormone release. Current recommendations suggest replacement of the potassium losses in diabetic ketoacidosis with the phosphate salt to compensate for depleted stores of 2,3-diphosphoglycerate. We caution physicians that such a regimen can result in severe electrolyte disturbances which potentially may be life threatening. Judicious use of potassium phosphate as an adjunct to traditional potassium chloride therapy, and close monitoring of serum calcium, magnesium and phosphorus, appears to be a preferable therapeutic regimen than potassium phosphate alone.

THE EFFECT OF 1,25-DIHYDROXYCHOLECALCIFEROL ON THE PARATHYROID HORMONE SECRETION OF PORCINE PARATHYROID GLANDS AND HUMAN PARATHYROID ADENOMAS IN VITRO

The effect of 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3) on parathyroid hormone secretion by porcine parathyroid glands and human parathyroid adenoma tissue was investigated by in vitro incubation. The addition of 100 nmoles 1,25-(OH)2-D3 to the medium inhibited significantly the release of immunoreactive parathyroid hormone by 63--65%. This suppression was reversible when 1,25-(OH)2-D3 was removed again. The inhibition of parathyroid hormone release observed in human parathyroid adenoma tissue was similar to that in normal porcine parathyroid glands. This indicates that adenoma tissue is sensitive to regulatory influences. As well as calcium, 1,25-(OH)2-D3 may act as another feedback inhibitor of parathyroid hormone secretion.