INTRACELLULAR SIGNALS THAT MEDIATE PTH SECRETION

The release of parathyroid hormone (PTH) by parathyroid glands is regulated by changes in extracellular calcium (Ca). High Ca suppresses PTH secretion and low Ca stimulates hormone release. The intracellular events that mediate the regulation of PTH secretion by Ca are becoming increasingly more understood. Ca modulates parathyroid function by acting on a G protein-coupled calcium-sensing receptor (CaR)^3,4. This effector system includes the hydrolysis of membrane phospholipids by phospholipase C (PLC), phospholipase D (PLD), and PLA₂ to generate the appropriate intracellular signals^5,6,7.

Activation of the CaR by high Ca triggers the activation of the Gq/11-protein-dependent PLC, causing the breakdown of phosphatidyl inositol 4,5 biphosphate with the generation of inositol triphosphate (IP3), diacylglycerol (DG), and phosphatidic acid (PA), and promoting a rapid elevation of cytosolic calcium, which is sustained by the release of calcium from intracellular stores^3,8. Both IP3 and DG serve as intracellular calcium messengers; PA is a substrate of PLA₂, a calcium-dependent lipase that releases free AA.

Bourdeau et al⁵ first investigated the involvement of PLA₂-derived AA and its metabolites in the regulation of PTH secretion. These authors showed in vitro that increasing extracellular calcium (Ca) concentrations increased free AA release and decreased PTH secretion, and that exogenous addition of PLA₂ and AA inhibited PTH secretion in a dose-dependent manner Figure 1. Bordeau et al^5,6 also showed that the downstream-oxygenated products of AA, 12-, and 15-HETES, which are produced through the 12- and 15-LO pathway, are potent inhibitors of PTH secretion. Furthermore, Kifor et al⁷ reported that the activation of the CaR by Ca results in a PKC-mediated activation of PLD to generate phosphatidylbutanol and PLA₂ to release AA. The pretreatment of parathyroid cells with PKC activators stimulated the release of AA at low and high Ca, while PKC inhibitors reduced AA release at high Ca to the level observed at low Ca alone.

Figure 1.

Effects of exogenous AA on porcine parathyroid cell secretion (A). Cells were incubated in vitro with 0.5 m Ca and different concentrations of AA for 90 minutes, and PTH release was determined. One hundred percent represents PTH secretion in cells incubated in 0.5 m Ca (3.42 0.22 pg/g protein), and 0% is PTH secretion in cells incubated in 2 m Ca (2.11 0.08 pg/g protein). Values are the mean SE of three experiments, assayed in triplicate. Effects of exogenous PLA₂ on porcine parathyroid cell secretion incubated in vitro with 0.5 m Ca, and different concentrations of PLA₂ for 90 minutes on PTH release was determined (B). One hundred percent represents PTH secretion in cells incubated in 0.5 m Ca (3.25 0.11 pg/g protein), and 0% is PTH secretion in cells incubated in 2 m Ca (1.88 0.08 pg/g protein). Values are the mean SE of four experiments, assayed in triplicate. Adapted from Bordeau et al⁵.

Full figure and legend (15K)

THE EFFECT OF HIGH PHOSPHATE ON AA PRODUCTION

Secondary hyperparathyroidism (HPT) is present in the majority of patients with acute and chronic renal failure. Phosphate (P) retention, in addition to hypocalcemia and a deficit of calcitriol, is an important factor involved in the pathogenesis of renal hyperparathyroidism. A major contribution to the knowledge of the role of AA on parathyroid gland function has come from the studying the direct effect of P on PTH secretion. Because a high extracellular phosphate (P) concentration prevents the normal inhibition of PTH secretion by high calcium^9,10,11, a number of in vitro studies were performed to determine whether AA was involved in the modulation of PTH secretion by high phosphate. In glands incubated with high extracellular P and Ca, the addition of AA to the culture resulted in the restoration of the normal inhibition of PTH secretion⁹. The measurement of AA production showed that the stimulatory effect of phosphate on PTH secretion was associated with a decrease in AA synthesis; however, the addition of exogenous PLA₂ to the same concentrations that induced a dose-dependent inhibition of PTH secretion in the low calcium medium⁵ did not reduce the increased PTH secretion induced by high phosphate¹².

REGULATION OF AA PRODUCTION BY INTRACELLULAR CALCIUM

In other cells, PLA₂ activity is dependent on intracellular calcium levels (Cai)¹³. In parathyroid cells the Cai level increases in response to the calcium receptor-dependent PLC activation^3,4,8. Thus, in a recent study we evaluated whether, in parathyroid cells, AA production is stimulated by an increase in Cai levels¹⁴. As shown in Figure 2, the elevation of Cai induced by both ionophore A23187 and thapsigargin, which increase the calcium influx across the cell membrane and through the release of calcium from intracellular stores, respectively^15,16, stimulated AA production and was associated with a decrease in PTH secretion. An inhibition of PTH secretion by the ionophore has been described by other authors^17,18,19; our results demonstrate that an increase in cytosolic calcium activates PLA₂, which suggests that PLC may stimulate PLA₂ via intracellular calcium increase. The precise mechanisms by which high intracellular calcium stimulates PLA₂ activity in parathyroid cells are not completely clear. It would be a direct effect, as PLA₂ is known to be a calcium-dependent enzyme. However, a recent work by Kifor et al²⁰ documented a role of the mitogen-activated protein (MAP) kinase cascade in the activation of the PLA₂ by phosphorylation. Their data suggest that the CaR activates the extracellular signal–regulated kinase 1 and 2 (ERK1/2) through protein kinase C (PKC) presumably through Gq/11-mediated activation of PI-PLC, as well as through G(i)- and protein tyrosine kinase (PTK)-dependent pathway(s), indicating the importance of the MAPK in PLA₂ activation. Furthermore, in a recent paper, Corbetta et al²¹ confirm the involvement of a CaR-mediated ERK1/2 activation, which participates in the inhibition of the PTH secretion by high extracellular Ca. This signaling pathway has shown to be PKC-dependent in normal parathyroid cells; however, it is very interesting that it is disrupted in parathyroid tumors: adenomatous cells showed high PKC-dependent ERK1/2 activity in resting conditions but were unresponsive to high extracellular Ca.

Figure 2.

AA production by dog parathyroid tissue incubated with high (A) or low (B) calcium (Ca) concentration. Experiments were performed with 1 or 4 m phosphate (P) concentration, with or without addition of 10 m ionophore (A23187) or 1 m thapsigargin. Values are mean SEM (N = 6 for the thapsigargin groups, N = 10 for the other groups). *P < 0.05 vs. all other columns in A. **P < 0.05 vs. ionophore and thapsigargin groups in B. Adapted from Almaden et al¹⁴.

Full figure and legend (58K)

Since AA production by parathyroid cells is decreased by high extracellular phosphate, we evaluated the effect of an elevation of the intracellular calcium concentration on AA production in the presence of high extracellular phosphate levels. The results Figure 2 showed that the elevation of the cytosolic calcium concentrations overcome the suppressive effect of high extracellular phosphate on AA production. Therefore, the reduced production of AA by high P seems to be related with a defective activation of PLA₂ because of an inability to generate the appropriate elevation in the cytosolic calcium levels. Further studies will be required to investigate how high phosphate levels affect the regulation of intracellular calcium levels. Reasonably, this mechanism should rely on a connection between the elevation of intracellular calcium and the activation of a kinase cascade involving the PKC and/or a MAPKinase pathway; alternatively, it would implicate a novel mechanism. Interestingly, the study by Quitterer et al²² on the signaling mechanisms responsible for the inhibitory effect of low magnesium (Mg) on PTH release has shown a novel mechanism of activation of the CaR. The Mg-binding site responsible of the inhibition of PTH secretion in vitro was different than that of calcium; Mg deficiency leads to a decrease in intracellular Mg, which increases the activity of Gi subunits of heterotrimeric G proteins.

In conclusion, AA appears to play a crucial role in the intracellular signaling mechanisms that regulate the parathyroid cell function. A high phosphate concentration decreases the production of AA by parathyroid tissue, so we could understand the physiopathologic features in the initiation or further development of the HPT secondary to renal failure by phosphate. The increased elucidation of the role of AA in parathyroid signaling may help to provide new insight in the understanding of the function of the parathyroid CaR, a preferred target for therapy in the secondary hyperparathyroidism.

References

1. Huang, S, Konieczkowski, M, Schelling, JR, Sedor, JR: Interleukin-1 stimulates Jun N-terminal/stress-activated protein kinase by an arachidonate-dependent mechanism in mesangial cells. Kidney Int 1999 55: 1740–1749,  | Article | PubMed | ISI | ChemPort |
2. Rizzo, MT, Carlo-Stella, C: Arachidonic acid mediates interleukin-1 tumor necrosis factor--induced activation of the c-jun amino-terminal kinases in stromal cells. Blood 1996 88: 3792–3800,  | PubMed | ISI | ChemPort |
3. Brown, EM, Gamba, G, Riccardi, D: Cloning and characterization of an extracellular Ca²⁺-sensing receptor from bovine parathyroid. Nat Lond 1993 366: 575–580,  | ChemPort |
4. Brown, EM, MacLeod, RJ: Extracellular calcium sensing and extracellular calcium signalling. Physiol Rev 2001 81(1):239–297,  | PubMed | ISI | ChemPort |
5. Bourdeau, A, Souberbielle, J-C, Bonnet, P, et al: Phospholipase A₂ action and arachidonic metabolism in calcium-mediated parathyroid hormone secretion. Endocrinolgy 1992 130: 1339–1344,  | ChemPort |
6. Bourdeau, A, Moutahir, M, Souberbielle, JC, et al: Effects of lipoxygenase products of arachidonate metabolism on parathyroid hormone secretion. Endocrinolgy 1994 135: 1109–1112,  | ChemPort |
7. Kifor, O, Diaz, R, Butters, R, Brown, EM: The Ca²⁺-sensing receptor (CaR) activates phospholipases C, A₂, and D in bovine parathyroid and CaR-transfected human embryonic kidney (HEK 293) cells. J Bone Miner Res 1997 12: 715–725,  | PubMed | ISI | ChemPort |
8. Brown, EM, Vassilev, PM, Quinn, S, Hebert, SC: G-protein-coupled, extracellular Ca²⁺ sensing receptor: A versatile regulator of diverse cellular functions. Vitam Horm 1999 55: 1–71,  | PubMed | ISI | ChemPort |
9. Almadén, Y, Canalejo, A, Hernández, A, et al: Direct effect of phosphorus on parathyroid hormone secretion from whole rat parathyroid glands in vitro. J Bone Miner Res 1996 11: 970–976,  | PubMed | ISI | ChemPort |
10. Almadén, Y, Hernández, A, Torregrosa, V, et al: High phosphate directly stimulates PTH secretion and synthesis by human parathyroid tissue. J Am Soc Nephrol 1998 9: 1845–1852,  | PubMed | ISI | ChemPort |
11. Hernández, A, Concepcion, MT, Rodríguez, M, et al: High phosphate diet increases prepro PTHmRNA independent of calcium and calcitriol in normal rats. Kidney Int 1996 50: 1872–1878,  | PubMed | ISI | ChemPort |
12. Almadén, Y, Canalejo, A, Ballesteros, E, et al: The effect of high extracellular phosphate concentration on arachidonic acid production by parathyroid tissue in vitro. J Am Soc Nephrol 2000 11: 1712–1718,  | PubMed | ISI | ChemPort |
13. Bonventre, JV: Phospholipase A₂ and signal transduction. J Am Soc Nephrol 1992 3: 128–150,  | PubMed | ISI | ChemPort |
14. Almadén, Y, Canalejo, A, Ballesteros, E, et al: Regulation of arachidonic acid production by intracellular calcium in parathyroid cells: Effect of extracellular phosphate. J Am Soc Nephrol 2002 13: 693–698,  | PubMed |
15. Thastrup, O, Cullemn, PJ, Drobak, BK, et al: Thapsigargin, a tumor promoter, discharges intracellular Ca²⁺ stores by specific inhibition of the endoplasmic reticulum Ca²⁺-ATP_ase. Proc Natl Acad Sci USA 1990 87: 2466–2470,  | PubMed | ChemPort |
16. Breitwiser, GE, Gama, L: Calcium-sensing receptor activation induces intracellular calcium oscillations. Am J Physiol 2001 280: C1412–C1421,
17. Habener, JF, Stevens, TD, Ravazzola, M, et al: Effects of calcium ionophores on the synthesis and release of parathyroid hormone. Endocrinology 1977 101: 1524–1537,  | PubMed | ISI | ChemPort |
18. Mihai, R, Lai, T, Schofield, GJ, Farndon, JR: Changes in cytoplasmic calcium determine the secretory response to extracellular cations in human parathyroid cells: A confocal microscopy study using FMI-43 dye. Biochem J 2000 352: 353–361,  | Article | PubMed | ISI | ChemPort |
19. Shoback, D, Chen, TH, Pratt, S, Lattyak, B: Thapsigargin stimulates intracellular calcium mobilization and inhibits parathyroid hormone release. J Bone Miner Res 1995 19: 743–750,
20. Kifor, O, MacLeod, RJ, Diaz, R, et al: Regulation of MAP Kinase by calcium-sensing receptor in bovine parathyroid and CaR transfected HEK293 cells. Am J Physiol Renal Physiol 2001 280: F291–F302,  | PubMed | ISI | ChemPort |
21. Corbetta, S, Lania, A, Filopanti, M, et al: Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells. J Clin Endocrinol Metab 2002 87(5):2201–2205,  | Article | PubMed | ISI | ChemPort |
22. Quitterer, U, Hoffman, M, Freichel, M, Lohse, MJ: Paradoxical block of parahormone secretion is mediated by increased activity of G alpha subunits. J Biol Chem 2001 276(9):6763–6769,  | Article | PubMed | ISI | ChemPort |

Acknowledgments

This work was supported in part by grants from the Ministry of Science and Technology (SAF2001-0350) and Junta de Andalucia (Expte 245). During the course of this work, Dr. Almaden was supported by the Ministry of Science and Technology (Ramon-Cajal Program).