Skip to main content

Advertisement

SpringerLink
Log in

The high affinity calcium inhibition of parathyroid adenylate cyclase is not calmodulin dependent

  • Laboratory Investigations
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Summary

To investigate the possible role of calmodulin in the calcium sensitivity of parathyroid adenylate cyclase (AC), the effect of the calmodulin inhibitor trifluoperazine hydrochloride (TFP) on the calcium sensitivity of forskolin-stimulated AC activity was investigated in membranes prepared from normal porcine parathyroid glands. TFP inhibited AC in a concentration-dependent manner, the IC50 being approximately 100 μM. The inhibition of the enzyme occurred at roughly the same concentration of TFP in the presence and absence of calcium. Another calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), also inhibited AC in a calcium-independent manner with a IC50 of approximately 200 μM. The pattern of calcium inhibition of AC was compared in membranes prewashed with either EGTA or 2 μM ionic calcium plus 100 μM TFP in an attempt to remove endogenous calmodulin. Neither treatment significantly altered the apparent affinities of the two previously reported calcium inhibition sites, nor did they alter the relative contribution of the individual calcium inhibition sites to the overall calcium inhibition. Inclusion of 100 μM TFP in the incubation mixture resulted in no change in the apparent affinities of the calcium inhibition site although it did result in a significant decrease in the relative contribution of the high affinity site (P<0.05). Addition of exogenous calmodulin (5–50 μg/ml) had no significant effect on AC. We conclude from these studies that the inhibition of parathyroid AC by calcium is independent of calmodulin and that this enzyme has intrinsic high sensitivity to calcium.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Habener JF, Rosenblatt M, Potts JT Jr (1984) Parathyroid hormone: biochemical aspects of biosynthesis, secretion, action and metabolism. Physiol Rev 64:985–1053

    PubMed  CAS  Google Scholar 

  2. Shoback D, Thatcher J, Leombruno R, Brown E (1983) Effects of extracellular Ca++ and Mg++ on cytosolic Ca++ and PTH release in dispersed bovine parathyroid cells. Endocrinology 113:424–426

    PubMed  CAS  Google Scholar 

  3. Shoback DM, Thatcher J, Leombruno R, Brown EM (1984) Relationship between parathyroid hormone secretion and cytosolic calcium concentration in dispersed bovine parathyroid cells. Proc Natl Acad Sci (USA) 81:3113–3117

    Article  CAS  Google Scholar 

  4. Dufresne LR, Gitelman HJ (1972) A possible role of adenyl cyclase in the regulation of parathyroid activity by calcium. In: Talmage RV, Munson PL (eds) Calcium, parathyroid hormone and the calcitonins. Amsterdam, Excerpta Medica, 202–206

    Google Scholar 

  5. Matsuzaki S, Dumont JE (1972) Effect of calcium ion on horse parathyroid gland adenyl cyclase. Biochim Biophys Acta 284:227–234

    PubMed  CAS  Google Scholar 

  6. Rodriguez HJ, Morrison A, Slatopolsky E, Klahr S (1973) Adenylate cyclase of human parathyroid gland. J Clin Endocrinol Metab 47:319–325

    Google Scholar 

  7. Bellorin-Font E, Martin KJ, Freitag JJ, Anderson C, Sicard G, Slatopolsky E, Klahr S (1981) Altered parathyroid adenylate cyclase kinetics in hyperfunctioning human parathyroid glands. J Clin Endocrinol Metab 52:499–507

    PubMed  CAS  Google Scholar 

  8. Ontjes DA, Mahaffee DD, Wells SA (1981) Adenylate cyclase activity in human parathyroid tissues: reduced sensitivity to suppression by calcium in parathyroid adenomas as compared with normal glands from normocalcemic subjects or noninvolved glands from hyperparathyroid subjects. Metabolism 30:406–411

    Article  PubMed  CAS  Google Scholar 

  9. Oldham SB, Molloy CT, Lipson LG (1984) Calcium inhibition of parathyroid adenylate cyclase. Endocrinology 114:207–214

    PubMed  CAS  Google Scholar 

  10. Willgoss D, Jacobi JM, deJersey J, Bartley PC, Lloyd HM (1980) Effect of calcium on cyclic nucleotide phosphodiesterase in parathyroid tissue. Biochem Biophys Res Commun 94:763–768

    Article  PubMed  CAS  Google Scholar 

  11. Brown EM (1980) Calcium-regulated phosphodiesterase in bovine parathyroid cells. Endocrinology 107:1998–2003

    Article  PubMed  CAS  Google Scholar 

  12. Pines M and Hurwitz S (1981) Cyclic AMP activates and calcium inhibits protein kinase activity in avian parathyroid glands. FEBS Lett 133:27–30

    Article  PubMed  CAS  Google Scholar 

  13. Brown EM, Thatcher JG (1982) cAMP-dependent protein kinase and the regulation of parathyroid hormone release by divalent cations and agents elevating cellular cAMP in dispersed bovine parathyroid cells. Endocrinology 110:1374–1380

    PubMed  CAS  Google Scholar 

  14. Cheung WY (1980) Calmodulin plays a pivotal role in cellular regulation, Science 207:19–26

    Article  PubMed  CAS  Google Scholar 

  15. Means AR, Tash JS, Chafouleas JG (1982) Physiological implications of the presence, distribution, and regulation of calmodulin in eukaryotic cells. Physiol Rev 62:1–39

    PubMed  CAS  Google Scholar 

  16. Brown EM, Dawson-Hughes BF, Wilson RE, Adragna N (1981) Calmodulin in dispersed human parathyroid cells. J Clin Endocrinol Metab 53:1064–1071

    PubMed  CAS  Google Scholar 

  17. Oldham SB, Lipson LG, Tietjen GE (1982) The presence of calmodulin in parathyroid adenomas. Mineral Electrolyte Metab 7:273–280

    CAS  Google Scholar 

  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  19. Bartfai T (1979) Preparation of metal-chelate complexes and the design of steady-state kinetic experiments involving metal nucleotide complexes. Adv Cyclic Nucleotide Res 10:219–242

    PubMed  CAS  Google Scholar 

  20. Salomon Y (1979) Adenylate cyclase assay. Adv Cyclic Nucleotide Res 10:35–55

    PubMed  CAS  Google Scholar 

  21. Dunn OJ, Clark VA (1974) Applied statistics: analysis of variance and regression. John Wiley and Sons, New York, p 94

    Google Scholar 

  22. Weiss B, Prozialeck W, Cimino M, Barnette MS, Wallace TL (1980) Pharmacological regulation of calmodulin. Ann NY Acad Sci 356:319–345

    Article  PubMed  CAS  Google Scholar 

  23. Hidaka H, Sasaki Y, Tanaka T, Endo T, Ohno S, Fujii Y, Nagata T (1981) N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad Sci (USA) 78:4354–4357

    Article  CAS  Google Scholar 

  24. Oldham SB, Rude RK, Molloy CT, Lipson LG (1984) The effects of magnesium on calcium inhibition of parathyroid adenylate cyclase. Endocrinology 115:1883–1890

    PubMed  CAS  Google Scholar 

  25. Wolff DJ, Poirier PG, Brostrom CO, Brostrom MA (1977) Divalent cation binding properties of bovine brain Ca2+-dependent regulator protein. J Biol Chem 252:4108–4117

    PubMed  CAS  Google Scholar 

  26. Oldham SB (1982) Calmodulin: its role in calcium-mediated cellular regulation. Mineral and Electrolyte Metab 8:1–12

    CAS  Google Scholar 

  27. Tanaka T, Ohmura T, Hidaka H (1982) Hydrophobic interaction of the Ca2+-calmodulin complex with calmodulin antagonists: naphthalenesulfonamide derivatives. Molec Pharmacol 22:403–407

    CAS  Google Scholar 

  28. Weiss B, Fertel R, Figlin R, Uzunov P (1974) Selective alteration of the activity of the multiple forms of adenosine 3′,5′-monophosphate phosphodiesterase of rat cerebrum. Mol Pharmacol 10:615–625

    CAS  Google Scholar 

  29. Levin RM, Weiss B (1979) Inhibition by trifluoperazine of calmodulin-induced activation of ATPase activity of rat erythrocyte. Neuropharmacology 19:169–174

    Article  Google Scholar 

  30. Schatzman RC, Wise BC, Kuo JF (1981) Phospholipid-sensitive calcium-dependent protein kinase: inhibition by antipsychotic drugs. Biochem Biophys Res Commun 98:669–676

    Article  PubMed  CAS  Google Scholar 

  31. Wise BC, Glass DB, Chou C-HJ, Raynor RL, Katoh N, Schatzman RC, Turner RS, Kibler RF, Kuo JF (1982) Phospholipid-sensitive Ca2+-dependent protein kinase from heart: II substrate specificity and inhibition by various agents. J Biol Chem 257:8489–8495

    PubMed  CAS  Google Scholar 

  32. Abe M, Sherwood LM (1972) Regulation of parathyroid hormone secretion by adenyl cyclase. Biochem Biophys Res Commun 48:396–401

    Article  PubMed  CAS  Google Scholar 

  33. Brown EM, Gardner DG, Windeck RA, Aurbach GD (1978) Relationship of intracellular 3′,5′-adenosine monophosphate accumulation to parathyroid hormone release from dispersed bovine parathyroid cells. Endocrinology 103:2323–2333

    PubMed  CAS  Google Scholar 

  34. Morrissey JJ, Cohn DV (1979) Regulation of secretion of parathormone and secretory protein-1 from separate intracellular pools by calcium, dibutyryl cyclic AMP, and (1)-isoproterenol. J Cell Biol 82:93–102

    Article  PubMed  CAS  Google Scholar 

  35. Shoback DM, Brown EM (1984) Forskolin increases cellular cyclic adenosine monophosphate content and parathyroid hormone release in dispersed bovine parathyroid cells. Metabolism 33:509–514

    Article  PubMed  CAS  Google Scholar 

  36. Hanley DA, Takatsuki K, Birnbaumer ME, Schneider AB, Sherwood LM (1980) In vitro perifusion for the study of parathyroid hormone secretion: effects of extracellular calcium concentration and beta-adrenergic regulation on bovine parathyroid hormone secretion in vitro. Calcif Tissue Int 32:19–27

    Article  PubMed  CAS  Google Scholar 

  37. Cantley LK, Scott DJ, Cooper CW, Mahaffee DD, Leight GS, Thomas CG, Ontjes DA (1984) Divergent effects of forskolin on 3′,5′ cyclic adenosine monophosphate production and parathyroid hormone secretion. Calcif Tissue Int 36:87–94

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medicine, University of Southern California School of Medicine, 2025 Zonal Avenue, 90033, Los Angeles, California

    Susan B. Oldham Ph.D. & Loren G. Lipson

Authors
  1. Susan B. Oldham Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Loren G. Lipson
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Oldham, S.B., Lipson, L.G. The high affinity calcium inhibition of parathyroid adenylate cyclase is not calmodulin dependent. Calcif Tissue Int 38, 275–281 (1986). https://doi.org/10.1007/BF02556606

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02556606

Key words

Search

Navigation