Dispersed Bovine Parathyroid Cells Research Articles

Low calcium decreases levels of vitamin D3 receptor (VDR) mRNA and inhibits homologous up-regulation of VDR mRNA BY 1, 25 (OH)2 D3 in dispersed bovine parathyroid cells

Low calcium decreases levels of vitamin D3 receptor (VDR) mRNA and inhibits homologous up-regulation of VDR mRNA BY 1, 25 (OH)2 D3 in dispersed bovine parathyroid cells

Bovine Parathyroid Glands Secrete a 26-kDa NTerminal Fragment of Chromogranin-A which Inhibits Parathyroid Cell Secretion*

Chromogranin-A (CgA) is a ubiquitous protein which colocalizes in secretory granules of multiple endocrine tissues and cosecretes with peptide hormones from these tissues. Although the function of CgA has remained unknown, there has been recent interest in its potential role as a prohormone for smaller, biologically active peptides. We isolated and characterized a 26-kDa N-terminal fragment of CgA which is a natural breakdown product of bovine parathyroid CgA in storage. A similar, if not identical, fragment of CgA is secreted by bovine parathyroid glands. The secreted fragment elutes on HPLC and migrates on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and acid-urea gels in the same position as the 26-kDa N-terminal fragment. When added to the incubation medium of dispersed bovine parathyroid cells, the 26-kDa N-terminal fragment of CgA inhibits the low calcium-stimulated secretion of both PTH and CgA. This N-terminal fragment is homologous to betagranin, which is a 21-kDa N-terminal fragment of CgA that is generated from CgA in rat insulin granules. Thus, a naturally occurring betagranin-like N-terminal fragment of bovine parathyroid CgA is not only secreted itself, but can inhibit the secretion of PTH and intact CgA by bovine parathyroid cells. The processing of intact CgA to fragments such as the N-terminal fragment that we describe may be important in the autocrine or paracrine regulation of secretion.

Polyarginine, polylysine, and protamine mimic the effects of high extracellular calcium concentrations on dispersed bovine parathyroid cells.

We investigated the effects of the basic peptides polyarginine, protamine, and polylysine on dispersed bovine parathyroid cells. All three peptides produced a dose-dependent inhibition of dopamine-stimulated cAMP accumulation, with half-maximal inhibition at 4 x 10(-8), 1.5 x 10(-7), 3 x 10(-7), and 2 x 10(-6) M, respectively, for polyarginine, protamine, and two preparations of polylysine of molecular weights 10,200 and 3800. The inhibition of cAMP accumulation was reversible and was blocked by preincubating the cells overnight with 0.5 micrograms/ml of pertussis toxin. The same peptides also inhibited PTH release at similar concentrations, markedly stimulated the accumulation of inositol phosphates at two- to threefold higher concentrations, and produced transient increases in the cytosolic Ca2+ concentration (Cai) in fura-2-loaded parathyroid cells. The polylysine-evoked spike in Cai persisted despite the removal of extracellular Ca2+, indicating that it arose from intracellular Ca2+ stores. Exposure of the cells to elevated extracellular magnesium (Mg2+) concentrations elicited a similar spike in Cai but blocked the Cai transient in response to subsequent addition of polylysine, or vice versa. Thus, Mg2+ and polylysine mobilize Ca2+ from the same intracellular store(s). These results indicate that highly basic peptides closely mimic the effects of polyvalent cations on parathyroid function, suggesting that both agents may regulate parathyroid function via similar biochemical pathways.

Neomycin mimics the effects of high extracellular calcium concentrations on parathyroid function in dispersed bovine parathyroid cells.

We examined the effects of the polycationic antibiotic, neomycin, on the function of dispersed bovine parathyroid cells. Neomycin caused a reversible, dose-dependent inhibition of low calcium (Ca++)-stimulated PTH release, with half-maximal inhibition at 30 microM. Maximal inhibition (with 200 microM neomycin) was not additive with the suppressive effects of high (2 mM) Ca++. Neomycin also inhibited dopamine-stimulated cAMP accumulation by 90-98% at 100-200 microM, with a half-maximal effect at 40-50 microM. This action was reversible and was blocked by preincubating the cells overnight with 0.5 microgram/ml pertussis toxin. In addition to its suppressive effects on cAMP metabolism and PTH release, neomycin stimulated the accumulation of inositol phosphates and produced a transient increase in the cytosolic Ca++ concentration (Cai) in fura-2-loaded parathyroid cells. The neomycin-evoked spike in Cai persisted despite removal of extracellular Ca++, indicating that it arises from intracellular Ca++ stores. Exposure of cells to elevated magnesium (Mg++) concentrations elicited a similar spike in Cai but blocked the spike in Cai in response to subsequent addition of neomycin and vice versa. Thus, Mg++ and neomycin mobilize Ca++ from the same intracellular store(s). These results indicate that a polycation, neomycin, closely mimics the effects of polyvalent cations on parathyroid function, suggesting that both agents regulate parathyroid function via similar biochemical pathways.

Differences in the Actions of Calcium Versus Lanthanum to Influence Parathyroid Hormone Release*

PTH release from bovine parathyroid cells is inhibited by increasing concentrations of extracellular calcium (Ca2+). We have proposed that this inhibition is mediated by Ca2+ channels via a G-protein. To further test this hypothesis, we evaluated the effect of lanthanum (La3+), a potent Ca2+ channel antagonist that does not cross the cell membrane. PTH release was determined in dispersed bovine parathyroid cells by radioimmunoassay: extracellular Ca2+ concentration was 0.2 mM. PTH release was inhibited by maximal concentrations of La3+ to a greater extent than by Ca2+: 93% inhibition by La3+ vs. 40% by Ca2+. La3+ was more potent (set-point = 0.12 mM) than Ca2+ (set-point = 1.2 mM). Incubation of parathyroid cells with pertussis toxin, which inactivates a G-protein(s) and blocks inhibition by Ca2+, did not block the inhibition of PTH release by La3+ at the concentrations tested. The Ca2+ ionophore A23187, which potentiates the effect of Ca2+, did not enhance the inhibition of PTH release by La3+. Increasing concentrations of calcium enhanced the inhibition of PTH release by the Ca2+ channel agonist, (+)202-791. The Ca2+ channel antagonist, (-)202-791, shifted the Ca2+ inhibition curve to the right. La3+ did not alter the inhibition of PTH release by the Ca2+ channel agonist but blocked the stimulatory effect of the Ca2+ channel antagonist, (-)202-791. In summary: 1) La3+, which blocks Ca2+ channels and does not cross cell membranes, effects a greater inhibition of PTH release than Ca2+; 2) La3+, like Ca2+, overrides the effect of Ca2+ channel antagonist (-)202-791; and 3) La3+, unlike Ca2+, inhibits PTH release by a mechanism that is independent of a pertussis toxin-sensitive G-protein. There may be two cell surface sites that recognize La3+ and Ca2+ independently.

The diltiazem analog TA-3090 mimics the actions of high extracellular Ca2+ on parathyroid function in dispersed bovine parathyroid cells

We previously showed that the calcium channel blocker diltiazem raises cytosolic Ca2+ and inhibits PTH release in bovine parathyroid cells. To investigate further possible mechanisms underlying these effects, we examined the effects of the more potent diltiazem analog TA-3090, which is a Ca2+ channel antagonist in vascular smooth muscle, on several aspects of the function of dispersed bovine parathyroid cells. Like diltiazem, TA-3090 (10(-6)-10(-4] produced a dose-dependent inhibition of immunoreactive PTH release at 0.5 mM Ca2+ and raised the cytosolic Ca2+ concentration by 25-50% in fura-2-loaded parathyroid cells in the presence but not in the absence of extracellular Ca2+, suggesting that it activated rather than inhibited Ca2+ channels. To determine whether this compound affects other aspects of parathyroid function, we examined its effects on the inhibition of cAMP accumulation by Ca2+, a process we recently found to involve inhibition of cAMP generation by Gi through a receptorlike mechanism, which is independent of changes in cytosolic Ca2+. TA-3090 (10(-4) M) inhibited dopamine-stimulated cAMP accumulation by up to 75% (from 663 to 166 fmol per 10(5) cells), with a higher apparent potency at greater extracellular Ca2+ concentrations. Moreover, the addition of 10(-4) M TA-3090 potentiated the inhibitory effects of both Ca2+ and Mg2+, decreasing the concentration of the divalent cation necessary to produce half-maximal inhibition of cAMP accumulation by about twofold. In the absence of extracellular Ca2+, however, TA-3090 had no effect on the stimulation of cAMP by dopamine or on the inhibition of dopamine-stimulated cAMP accumulation by PGF2 alpha, which also regulates cAMP via Gi.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Cytosolic pH in Bovine Parathyroid Cells: Effect of Fluoride*

In the present investigation, sodium fluoride (NaF) was employed to explore the role of guanine nucleotide-binding proteins (G-proteins), protein kinase-C, or cytosolic calcium [( Ca]i) in the regulation of cytosolic pH [( pH]i) in dispersed bovine parathyroid cells, using the pH-sensitive fluorescent dye BCECF. When cells acidified by nigericin in Na-free medium were resuspended in Na-containing buffer, [pH]i returned to basal levels. This recovery was blocked by continued removal of Na+ or the addition of amiloride. NaF (10 mM) increased [32P]phosphate incorporation into phosphatidylinositol bisphosphate, suggesting an increase in phosphatidylinositol bisphosphate turnover. NaF caused an initial acidification, followed by an alkaline recovery in a dose-dependent manner (1-10 mM). Amiloride blocked the NaF-induced alkaline recovery. The protein kinase-C activator phorbol 12-myristate 13-acetate (10(-7) M) caused cytosolic alkalinization, while the protein kinase-C inhibitor H7 (6 x 10(-5) M) significantly inhibited the NaF-induced alkaline recovery. Pertussis toxin (1 microgram/ml) did not affect the NaF-induced changes in [pH]i. Removal of extracellular Ca2+ with EGTA blocked the NaF-induced increase in [Ca]i and alkaline recovery. Ionomycin (5 x 10(-7) M) caused cytosolic alkalinization, but pretreatment with EGTA inhibited the ionomycin-induced cytosolic alkalinization. The present studies clearly demonstrated the presence of an amiloride-sensitive Na+/H+ exchanger in parathyroid cells. Our findings suggest that the NaF-induced cytosolic alkaline recovery was via two complementing pathways: 1) activation of protein kinase-C, followed by stimulation of a Na+/H+ exchanger, and 2) existence of extracellular calcium and/or an increase in [Ca]i.

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.

Direct demonstration of D1 dopamine receptors in the bovine parathyroid gland using the D1 selective antagonist [125I]‐SCH 23982

The presence of D1 dopamine receptors in the parathyroid gland has been proposed based on the demonstration of dopaminergic regulation of adenylate cyclase activity and parathyroid hormone release in dispersed bovine parathyroid cells. Using a radioiodinated D1 selective antagonist [125I]-SCH 23982, we have now directly labeled and characterized the D1 dopamine receptors in bovine parathyroid gland membranes. [125I]-SCH 23982 binds in a saturable manner with high affinity and low nonspecific binding to membranes prepared from bovine parathyroid glands. D1 dopamine receptors are present in this preparation at a concentration of approximately 130 fMoles/mg protein and [125I]-SCH 23982 binding increases with increasing protein concentration in a linear fashion. Determination of the Kd using the association (k1) and dissociation (k-1) rate constants revealed good agreement with the Kd determined by saturation analysis (390 pM vs. 682 pM, respectively). Inhibition of 0.3 nM [125I]-SCH 23982 binding by a series of dopaminergic antagonists verified the D1 nature of this binding site, exhibiting appropriate affinities and rank order of potency. The competition curves of all antagonists exhibited Hill coefficients that were not significantly different from 1. Inhibition of [125I]-SCH 23982 binding by dopamine and other dopaminergic agonists revealed the presence of high and low affinity agonist binding sites. Addition of 200 microM GppNHp effected a complete conversion of high affinity dopamine binding sites to a homogeneous population of low affinity dopamine sites. The D1 receptors identified in the parathyroid gland with [125I]-SCH 23982 appear to be pharmacologically identical with those previously characterized in the central nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of hormone secretion and cytosolic Ca 2+ by extracellular Ca 2+ in parathyroid cells and C-cells: Role of voltage-sensitive Ca 2+ channels

The two dihydropyridine enantiomers, (+)202-791 and (−)202-791, that act as voltage-sensitive Ca 2+ channel agonist and antagonist, respectively, were examined for effects on cytosolic Ca 2+ concentrations ([Ca 2+] i) and on hormone secretion in dispersed bovine parathyroid cells and a rat medullary thyroid carcinoma (rMTC) cell line. In both cell types, small increases in the concentration of extracellular Ca 2+ evoked transient followed by sustained increases in [Ca 2+] i, as measured with fura-2. Increases in [Ca 2+] i obtained by raised extracellular Ca 2+ were associated with a stimulation of secretion of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in rMTC cells, but an inhibition of secretion of parathyroid hormone (PTH) in parathyroid cells. The Ca 2+ channel agonist (+)202-791 stimulated whereas the antagonist (−)202-791 inhibited both transient and sustained increases in [Ca 2+] i induced by extracellular Ca 2+ in rMTC cells. Secretion of CT and CGRP was correspondingly enhanced and depressed by (+)202-791 and (−)202-791, respectively. In contrast, neither the agonist nor the antagonist affected [Ca 2+] i and PTH secretion in parathyroid cells. Depolarizing concentrations of extracellular K + increased [Ca 2+] i and hormone secretion in rMTC cells and both these responses were potentiated or inhibited by the Ca 2+ channel agonist or antagonist, respectively. The results suggest a major role of voltage-sensitive Ca 2+ influx in the regulation of cytosolic Ca 2+ and hormone secretion in rMTC cells. Parathyroid cells, on the other hand, appear to lack voltage-sensitive Ca 2+ influx pathways and regulate PTH secretion by some alternative mechanism.

Fluoride stimulates the accumulation of inositol phosphates, increases intracellular free calcium, and inhibits parathyroid hormone release in dispersed bovine parathyroid cells.

The stimulation of polyphosphoinositide (PPI) turnover is associated with cellular activation and hormone secretion in numerous systems. GTP-binding proteins appear to couple receptors to phospholipase-C-mediated PPI breakdown. We assessed the effects of fluoride, an activator of GTP-binding proteins, on inositol phosphate accumulation, intracellular free Ca2+ [(Ca2+)i], cAMP content, and PTH release in dispersed bovine parathyroid cells. Sodium fluoride (5-30 mM) produced marked dose-dependent increases in inositol phosphates. With anion exchange HPLC, we confirmed that 30 mM fluoride stimulated a rapid increase in 1,4,5-inositol trisphosphate, a potent Ca2+-mobilizing compound. Using the Ca2+-sensitive probe fura-2, we determined that 30 mM fluoride increased [Ca2+]i from 339 +/- 9 to 650 +/- 39 nM (n = 8) within 30-60 sec at 1 mM extracellular Ca2+. After the depletion of extracellular Ca2+ by the addition of 1 mM EGTA, 30 mM fluoride increased [Ca2+]i 45 +/- 9% (n = 4), indicating that fluoride can mobilize intracellular Ca2+ stores. Fluoride (1-30 mM) also inhibited PTH release in dose-dependent fashion. Fluoride (30 mM) produced 72.8 +/- 4.2% suppression of maximal low Ca2+-stimulated PTH release comparable to the 83.7 +/- 3.7% inhibition by 2.0 mM extracellular Ca2+. Since changes in both [Ca2+]i and cAMP regulate PTH release, we measured the effect of fluoride on intracellular cAMP. Fluoride did not detectably change basal cAMP content, but it reduced forskolin-stimulated increases in cAMP. We conclude that fluoride may activate at least two GTP-dependent processes in parathyroid cells, resulting in PPI breakdown and cAMP accumulation. While both may contribute to the fluoride-induced suppression of PTH release, our findings suggest that the stimulation of PPI turnover leads to inhibition of PTH secretion.

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.

Effect of 1,25-dihydroxyvitamin D3 on cytosolic calcium in dispersed parathyroid cells

We examined the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on cytosolic calcium ([Ca]i) of dispersed bovine parathyroid cells, using the fluorescent dye indo-1. The addition of 10(-8) M 1,25-(OH)2D3 caused an increase in [Ca]i by 23.4 +/- 2.7% over a 10 minute period. There was a significant increase in [Ca]i within two minutes of the addition of 1,25-(OH)2D3. 1,25-(OH)2D3 increased [Ca]i in a dose-dependent manner and this occurred with as little as 10(-10) M. Neither 10(-7) M 25-(OH)D3 nor 10(-7) M 24, 25-(OH)2D3 caused a significant increase in [Ca]i. Chelation of extracellular calcium with EGTA blocked the 1,25-(OH)2D3-induced increase in [Ca]i, suggesting that the increase was mainly from extracellular calcium. Neither 10(-5) M verapamil nor 10(-4) M diltiazem blocked the 1,25-(OH)2D3-induced increase in [Ca]i. The present data suggest that 1,25-(OH)2D3 might modify membrane permeability to calcium independent of voltage-dependent calcium channels sensitive to verapamil or diltiazem. The rapid effect of 1,25-(OH)2D3 raises the possibility that its mechanism is independent of genome activation, perhaps attributable to direct interaction with components of the parathyroid cell plasma membrane.

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.

Effects of extracellular calcium and magnesium on cytosolic calcium concentration in Fura-2-loaded bovine parathyroid cells.

A newly developed calcium-sensitive dye, Fura-2, was employed in dispersed bovine parathyroid cells to study the effects of extracellular calcium and magnesium on cytosolic calcium concentration and parathyroid hormone (PTH) release. In comparison with control cells, Fura-2-loaded parathyroid cells showed the same maximal rate of PTH release, set-point for extracellular Ca++ (the calcium concentration producing half of the maximal inhibition of PTH release), and maximal inhibition of PTH release (71.6%) by high extracellular Ca++. At an extracellular Mg++ concentration of 0.5 mM, raising extracellular Ca++ in a stepwise fashion from 0.5 mM to 2.0 mM produced a dose-dependent, statistically significant (p less than 0.01) increase in cytosolic Ca++ from 198 +/- 24 nM (0.5 mM Ca++) to 411 +/- 21 nM (2.0 mM Ca++) which closely paralleled the concomitant decrease in PTH release. An elevation of extracellular Mg++ from 0.5 mM to 5 mM, at an extracellular Ca++ of 0.5 mM, resulted in a transient spike of cytosolic Ca++ which lasted for approximately 30 seconds, followed by a small but stable increase in the cytosolic Ca++ concentration (174 +/- 7 nM vs. 237 +/- 10 nM, n = 4, p less than 0.01). Prior removal of extracellular calcium by addition of an excess of EGTA did not abolish the transient spike induced by high extracellular magnesium concentrations in Fura-2-loaded cells, suggesting that this rapid increase in cytosolic Ca++ arises, at least in part, from intracellular stores of Ca++. This is supported by the observation that pretreating cells with ionomycin resulted in disappearance of the magnesium-induced spike.(ABSTRACT TRUNCATED AT 250 WORDS)

WR-2721 inhibits parathyroid adenylate cyclase.

WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] is a chemoprotective and radioprotective agent that has been shown to lower serum calcium in dogs and in humans. This is secondary both to impaired release of Ca2+ from bone and diminished secretion of parathyroid hormone (PTH) from parathyroid glands. Because cAMP plays a role in the regulation of PTH secretion and WR-2721 has been shown to lower cAMP levels in radiated mouse spleen, we investigated the effects of WR-2721 on cAMP production in dispersed bovine parathyroid cells. Additionally, we studied the adenylate cyclase in plasma membranes from normal bovine parathyroid glands after exposure to WR-2721. With parathyroid cells incubated at 0.5 mM Ca2+, addition of WR-2721 in concentrations ranging from 0.02 to 2.0 mM resulted in a progressive decrease in intracellular cAMP (42-50%, respectively). In plasma membranes of bovine parathyroid cells a dose-dependent decrease in adenylate cyclase activity was noted. Inhibition of the cyclase was seen over a wide range of Mg2+ concentrations (2.5-40 mM). WR-2721 inhibited both basal and NaF, Gpp(NH)p, forskolin, and pertussin toxin-stimulated adenylate cyclase. These data suggest that WR-2721 inhibits the activity of parathyroid adenylate cyclase.

Prostaglandin F2 alpha and alpha-adrenergic agonists regulate parathyroid cell function via the inhibitory guanine nucleotide regulatory protein.

Prostaglandin F2 alpha (PGF2 alpha) and alpha-adrenergic agonists inhibit cAMP production and PTH secretion in dispersed bovine parathyroid cells. We have tested the mechanism of these effects utilizing pertussis toxin which catalyzes ADP ribosylation and inactivation of the inhibitory adenylate cyclase coupling protein Ni. Dispersed bovine parathyroid cells treated with or without 0.5 micrograms/ml pertussis toxin were tested with stimulatory (epinephrine, isoproterenol) or inhibitory (PGF2 alpha) agonists for responses in cAMP accumulation (5-min incubation) or PTH (90-min incubation) release. Pertussis toxin produced an enhanced response to epinephrine (a mixed alpha-adrenergic and beta-adrenergic agonist) in cAMP production and in PTH secretion. PGF2 alpha inhibited intracellular cAMP by 40% and PTH secretion by 35%. Pertussis toxin treatment of bovine parathyroid cells reduced the PGF2 alpha inhibition. We conclude that: 1) inhibition of PTH release by PGF2 alpha and alpha-adrenergic agonists parallels inhibition of cAMP production; 2) pertussis toxin blocks the inhibitory actions of PGF2 alpha and alpha-adrenergic agents on cAMP accumulation and PTH secretion; 3) the inhibitory coupling protein Ni mediates the inhibitory effects of these agents.

Involvement of glutathione oxidation reduction in parathyroid hormone secretion

This study determines whether calcium affects glutathione metabolism and whether glutathione metabolism may influence parathyroid (PTH) secretion in collagenase dispersed bovine parathyroid cells. Reduced glutathione (GSH) and glutathione disulfide (GSSG) were measured fluorometrically and enzymatically while PTH secretion was determined by radioimmunoassay. The total GSH and GSSG content of parathyroid cells was found to range from 1.59 to 1.71 micrograms/mg cell protein, and this did not vary significantly with changes in extracellular calcium. An increase in the medium calcium concentration from 0.5 to 2.0 mM did, however, cause an increase in GSSG from 0.43-0.54 to 1.19-1.20 micrograms/mg protein with a concomitant decrease in GSH. The compound 2-cyclohexen-1-one was used to deplete the cells of GSH at a low-calcium medium (0.5 mM) to levels seen in high-calcium medium (2.0 mM). This treatment was found to inhibit PTH secretion in the low-calcium medium, as if the cells were incubated in high medium calcium. Both 2-cyclohexen-1-one and calcium caused a rapid decrease in reduced GSH levels and in hormone secretion. The ketone was not found to affect cellular protein synthesis, indicating that there was no nonspecific toxic effect of this treatment on the cells. These results suggest that changes in the calcium concentration of the medium affect the GSH/GSSG ratio of dispersed parathyroid cells. Changes in the GSH/GSSG ratio induced by calcium may be related to changes in PTH secretion.

Stimulation of parathyroid hormone secretion by phorbol esters is associated with a decrease of cytosolic calcium

Unlike in other endocrine systems calcium inhibits parathyroid hormone (PTH) secretion and this inhibition is paralleled by a rise of cytosolic calcium concentration ([Ca] i). Because of evidence that diglyceride levels and protein kinase C activity are also decreased by high extracellular calcium we have investigated the effects of 12- O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, on [Ca] i and PTH secretion using dispersed bovine parathyroid cells. At 1.5 mM medium calcium TPA enhanced PTH secretion and caused reduction of [Ca] i from 639 ± 36 nM (SE) to 335 ± 21 nM ( P < 0.001); at 0.5 mM calcium TPA was ineffective. Moreover, TPA suppressed the rise of [Ca] i evoked by high extracellular calcium. Thus TPA presumably stimulates PTH secretion via activation of protein kinase C, and the lowering of [Ca] i may be a secondary event related to diglyceride availability.

Modulation of intracellular Ca 2+ in the parathyroid cell : Release of Ca 2+ from non-mitochondrial pools by inositol trisphosphate

Stimuli which enhance secretion from parathyroid cells such as low extracellular Ca 2+ or Mg 2+ are associated with a decrease in the cytosolic Ca 2+ concentration as measured by quin2. Current evidence suggests that increased production of inositol 1,4,5-triphosphate (IP 3) releases Ca 2+ from cellular stores thus increasing cytosolic Ca 2+. We used saponin-permeabilized dispersed bovine parathyroid cells to study the effect of IP 3 on intracellular Ca 2+. IP 3 released Ca 2+ from these cells in a dose-dependent manner; half-maximal response occurred with 0.3 μM IP 3 and maximal response with 1.2 μM IP 3. Permeabilized cells incubated in the presence of the mitochondrial inhibitor antimycin A released a similar amount of Ca 2+ suggesting that IP 3 releases Ca 2+ from a non-mitochondrial pool. These results suggest that IP 3 regulates cytosolic Ca 2+ in this system and may function as a second messenger controlling hormone secretion.