Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Jul 1;90(13):6265–6269. doi: 10.1073/pnas.90.13.6265

Gi2 and protein kinase C are required for thyrotropin-releasing hormone-induced stimulation of voltage-dependent Ca2+ channels in rat pituitary GH3 cells.

M Gollasch 1, C Kleuss 1, J Hescheler 1, B Wittig 1, G Schultz 1
PMCID: PMC46909  PMID: 8392194

Abstract

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) and other secretion-stimulating hormones trigger an increase in the cytosolic Ca2+ concentration by two mechanisms. Ca2+ is released from intracellular stores in response to inositol 1,4,5-trisphosphate and can enter the cell through voltage-dependent L-type Ca2+ channels. Stimulation of these channels is sensitive to pertussis toxin, indicating that a pertussis toxin-sensitive heterotrimeric guanine nucleotide-binding regulatory protein (G protein) is involved in functional coupling of the receptor to the Ca2+ channel. We identified the G protein involved in the stimulatory effect of TRH on the Ca2+ channel by type-selective suppression of G-protein synthesis. Antisense oligonucleotides were microinjected into GH3 cell nuclei, and 48 h after injection the TRH effect was tested. Whereas antisense oligonucleotides hybridizing to the mRNA of G(o) or Gi1 alpha-subunit sequences did not affect stimulation by TRH, oligonucleotides suppressing the expression of the Gi2 alpha subunit abolished this effect, and oligonucleotides directed against the mRNA of the Gi3 alpha subunit had less effect. The requirement of a concurrent inositol phospholipid degradation and subsequent protein kinase C (PKC) activation for the TRH effect on Ca(2+)-channel activity was demonstrated by inhibitory effects of antisense oligonucleotides directed against Gq/G11/Gz alpha-subunit sequences and treatment of GH3 cells with PKC inhibitors, respectively. Our results suggest that TRH elevates the cytosolic Ca2+ concentration in GH3 cells transiently via Ca2+ release from internal stores, followed by a phase of sustained Ca2+ influx through voltage-dependent Ca2+ channels stimulated by the concerted action of Gi2 (and Gi3) plus PKC.

Full text

PDF
6265

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albert P. R., Tashjian A. H., Jr Relationship of thyrotropin-releasing hormone-induced spike and plateau phases in cytosolic free Ca2+ concentrations to hormone secretion. Selective blockade using ionomycin and nifedipine. J Biol Chem. 1984 Dec 25;259(24):15350–15363. [PubMed] [Google Scholar]
  2. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  3. Berstein G., Blank J. L., Smrcka A. V., Higashijima T., Sternweis P. C., Exton J. H., Ross E. M. Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1. J Biol Chem. 1992 Apr 25;267(12):8081–8088. [PubMed] [Google Scholar]
  4. Birnbaumer L., Abramowitz J., Brown A. M. Receptor-effector coupling by G proteins. Biochim Biophys Acta. 1990 May 7;1031(2):163–224. doi: 10.1016/0304-4157(90)90007-y. [DOI] [PubMed] [Google Scholar]
  5. Cantiello H. F., Patenaude C. R., Ausiello D. A. G protein subunit, alpha i-3, activates a pertussis toxin-sensitive Na+ channel from the epithelial cell line, A6. J Biol Chem. 1989 Dec 15;264(35):20867–20870. [PubMed] [Google Scholar]
  6. Cohen C. J., McCarthy R. T., Barrett P. Q., Rasmussen H. Ca channels in adrenal glomerulosa cells: K+ and angiotensin II increase T-type Ca channel current. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2412–2416. doi: 10.1073/pnas.85.7.2412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Di Virgilio F., Pozzan T., Wollheim C. B., Vicentini L. M., Meldolesi J. Tumor promoter phorbol myristate acetate inhibits Ca2+ influx through voltage-gated Ca2+ channels in two secretory cell lines, PC12 and RINm5F. J Biol Chem. 1986 Jan 5;261(1):32–35. [PubMed] [Google Scholar]
  8. Doupnik C. A., Pun R. Y. Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells. Pflugers Arch. 1992 Jan;420(1):61–71. doi: 10.1007/BF00378642. [DOI] [PubMed] [Google Scholar]
  9. Dufy B., Jaken S., Barker J. L. Intracellular Ca2+-dependent protein kinase C activation mimics delayed effects of thyrotropin-releasing hormone on clonal pituitary cell excitability. Endocrinology. 1987 Aug;121(2):793–802. doi: 10.1210/endo-121-2-793. [DOI] [PubMed] [Google Scholar]
  10. Fong H. K., Yoshimoto K. K., Eversole-Cire P., Simon M. I. Identification of a GTP-binding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin. Proc Natl Acad Sci U S A. 1988 May;85(9):3066–3070. doi: 10.1073/pnas.85.9.3066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gollasch M., Haller H., Schultz G., Hescheler J. Thyrotropin-releasing hormone induces opposite effects on Ca2+ channel currents in pituitary cells by two pathways. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10262–10266. doi: 10.1073/pnas.88.22.10262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gollasch M., Hescheler J., Spicher K., Klinz F. J., Schultz G., Rosenthal W. Inhibition of Ca2+ channels via alpha 2-adrenergic and muscarinic receptors in pheochromocytoma (PC-12) cells. Am J Physiol. 1991 Jun;260(6 Pt 1):C1282–C1289. doi: 10.1152/ajpcell.1991.260.6.C1282. [DOI] [PubMed] [Google Scholar]
  13. Gordeladze J. O., Sletholt K., Thorn N. A., Gautvik K. M. Hormone-sensitive adenylate cyclase of prolactin-producing rat pituitary adenoma (GH4C1) cells: molecular organization. Eur J Biochem. 1988 Nov 15;177(3):665–672. doi: 10.1111/j.1432-1033.1988.tb14421.x. [DOI] [PubMed] [Google Scholar]
  14. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  15. Hescheler J., Rosenthal W., Hinsch K. D., Wulfern M., Trautwein W., Schultz G. Angiotensin II-induced stimulation of voltage-dependent Ca2+ currents in an adrenal cortical cell line. EMBO J. 1988 Mar;7(3):619–624. doi: 10.1002/j.1460-2075.1988.tb02855.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hsu W. H., Rudolph U., Sanford J., Bertrand P., Olate J., Nelson C., Moss L. G., Boyd A. E., Codina J., Birnbaumer L. Molecular cloning of a novel splice variant of the alpha subunit of the mammalian Go protein. J Biol Chem. 1990 Jul 5;265(19):11220–11226. [PubMed] [Google Scholar]
  17. Imai A., Gershengorn M. C. Phosphatidylinositol 4,5-bisphosphate turnover is transient while phosphatidylinositol turnover is persistent in thyrotropin-releasing hormone-stimulated rat pituitary cells. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8540–8544. doi: 10.1073/pnas.83.22.8540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones D. T., Reed R. R. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem. 1987 Oct 15;262(29):14241–14249. [PubMed] [Google Scholar]
  19. Katada T., Gilman A. G., Watanabe Y., Bauer S., Jakobs K. H. Protein kinase C phosphorylates the inhibitory guanine-nucleotide-binding regulatory component and apparently suppresses its function in hormonal inhibition of adenylate cyclase. Eur J Biochem. 1985 Sep 2;151(2):431–437. doi: 10.1111/j.1432-1033.1985.tb09120.x. [DOI] [PubMed] [Google Scholar]
  20. Kiley S. C., Parker P. J., Fabbro D., Jaken S. Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4C1 cells. J Biol Chem. 1991 Dec 15;266(35):23761–23768. [PubMed] [Google Scholar]
  21. Kleuss C., Hescheler J., Ewel C., Rosenthal W., Schultz G., Wittig B. Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents. Nature. 1991 Sep 5;353(6339):43–48. doi: 10.1038/353043a0. [DOI] [PubMed] [Google Scholar]
  22. Kleuss C., Scherübl H., Hescheler J., Schultz G., Wittig B. Different beta-subunits determine G-protein interaction with transmembrane receptors. Nature. 1992 Jul 30;358(6385):424–426. doi: 10.1038/358424a0. [DOI] [PubMed] [Google Scholar]
  23. Kuan S. I., Login I. S., Judd A. M., MacLeod R. M. A comparison of the concentration-dependent actions of thyrotropin-releasing hormone, angiotensin II, bradykinin, and Lys-bradykinin on cytosolic free calcium dynamics in rat anterior pituitary cells: selective effects of dopamine. Endocrinology. 1990 Oct;127(4):1841–1848. doi: 10.1210/endo-127-4-1841. [DOI] [PubMed] [Google Scholar]
  24. Lewis D. L., Weight F. F. The protein kinase C activator 1-oleoyl-2-acetylglycerol inhibits voltage-dependent Ca2+ current in the pituitary cell line AtT-20. Neuroendocrinology. 1988 Feb;47(2):169–175. doi: 10.1159/000124909. [DOI] [PubMed] [Google Scholar]
  25. Marchetti C., Brown A. M. Protein kinase activator 1-oleoyl-2-acetyl-sn-glycerol inhibits two types of calcium currents in GH3 cells. Am J Physiol. 1988 Jan;254(1 Pt 1):C206–C210. doi: 10.1152/ajpcell.1988.254.1.C206. [DOI] [PubMed] [Google Scholar]
  26. McClue S. J., Selzer E., Freissmuth M., Milligan G. Gi3 does not contribute to the inhibition of adenylate cyclase when stimulation of an alpha 2-adrenergic receptor causes activation of both Gi2 and Gi3. Biochem J. 1992 Jun 1;284(Pt 2):565–568. doi: 10.1042/bj2840565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mollard P., Dufy B., Vacher P., Barker J. L., Schlegel W. Thyrotropin-releasing hormone activates a [Ca2+]i-dependent K+ current in GH3 pituitary cells via Ins(1,4,5)P3-sensitive and Ins(1,4,5)P3-insensitive mechanisms. Biochem J. 1990 Jun 1;268(2):345–352. doi: 10.1042/bj2680345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mollard P., Vacher P., Rogawski M. A., Dufy B. Vasopressin enhances a calcium current in human ACTH-secreting pituitary adenoma cells. FASEB J. 1988 Oct;2(13):2907–2912. doi: 10.1096/fasebj.2.13.2844618. [DOI] [PubMed] [Google Scholar]
  29. Offermanns S., Gollasch M., Hescheler J., Spicher K., Schmidt A., Schultz G., Rosenthal W. Inhibition of voltage-dependent Ca2+ currents and activation of pertussis toxin-sensitive G-proteins via muscarinic receptors in GH3 cells. Mol Endocrinol. 1991 Jul;5(7):995–1002. doi: 10.1210/mend-5-7-995. [DOI] [PubMed] [Google Scholar]
  30. Offermanns S., Schultz G., Rosenthal W. Secretion-stimulating and secretion-inhibiting hormones stimulate high-affinity pertussis-toxin-sensitive GTPases in membranes of a pituitary cell line. Eur J Biochem. 1989 Mar 15;180(2):283–287. doi: 10.1111/j.1432-1033.1989.tb14645.x. [DOI] [PubMed] [Google Scholar]
  31. Paulssen E. J., Paulssen R. H., Haugen T. B., Gautvik K. M., Gordeladze J. O. Cell specific distribution of guanine nucleotide-binding regulatory proteins in rat pituitary tumour cell lines. Mol Cell Endocrinol. 1991 Apr;76(1-3):45–53. doi: 10.1016/0303-7207(91)90258-t. [DOI] [PubMed] [Google Scholar]
  32. Rosenthal W., Hescheler J., Hinsch K. D., Spicher K., Trautwein W., Schultz G. Cyclic AMP-independent, dual regulation of voltage-dependent Ca2+ currents by LHRH and somatostatin in a pituitary cell line. EMBO J. 1988 Jun;7(6):1627–1633. doi: 10.1002/j.1460-2075.1988.tb02989.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shorte S. L., Schofield J. G. Thyrotropin-releasing hormone-induced cytosolic calcium transients: characterisation of store refilling in bovine anterior pituitary cells. Mol Cell Endocrinol. 1991 Aug;79(1-3):167–176. doi: 10.1016/0303-7207(91)90107-4. [DOI] [PubMed] [Google Scholar]
  34. Simonds W. F., Goldsmith P. K., Codina J., Unson C. G., Spiegel A. M. Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7809–7813. doi: 10.1073/pnas.86.20.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stojilković S. S., Iida T., Virmani M. A., Izumi S., Rojas E., Catt K. J. Dependence of hormone secretion on activation-inactivation kinetics of voltage-sensitive Ca2+ channels in pituitary gonadotrophs. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8855–8859. doi: 10.1073/pnas.87.22.8855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stojilković S. S., Torsello A., Iida T., Rojas E., Catt K. J. Calcium signaling and secretory responses in agonist-stimulated pituitary gonadotrophs. J Steroid Biochem Mol Biol. 1992 Mar;41(3-8):453–467. doi: 10.1016/0960-0760(92)90371-o. [DOI] [PubMed] [Google Scholar]
  37. Strathmann M., Simon M. I. G protein diversity: a distinct class of alpha subunits is present in vertebrates and invertebrates. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9113–9117. doi: 10.1073/pnas.87.23.9113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Suzuki N., Takagi H., Yoshioka T., Tanakadate A., Kano M. Augmentation of transient low-threshold Ca2+ current induced by GTP-binding protein signal transduction system in GH3 pituitary cells. Biochem Biophys Res Commun. 1992 Aug 31;187(1):529–536. doi: 10.1016/s0006-291x(05)81526-8. [DOI] [PubMed] [Google Scholar]
  39. Tallent M., Reisine T. Gi alpha 1 selectively couples somatostatin receptors to adenylyl cyclase in pituitary-derived AtT-20 cells. Mol Pharmacol. 1992 Mar;41(3):452–455. [PubMed] [Google Scholar]
  40. Tamaoki T. Use and specificity of staurosporine, UCN-01, and calphostin C as protein kinase inhibitors. Methods Enzymol. 1991;201:340–347. doi: 10.1016/0076-6879(91)01030-6. [DOI] [PubMed] [Google Scholar]
  41. Thorn P., Petersen O. H. Activation of voltage-sensitive Ca2+ currents by vasopressin in an insulin-secreting cell line. J Membr Biol. 1991 Oct;124(1):63–71. doi: 10.1007/BF01871365. [DOI] [PubMed] [Google Scholar]
  42. Trautwein W., Hescheler J. Regulation of cardiac L-type calcium current by phosphorylation and G proteins. Annu Rev Physiol. 1990;52:257–274. doi: 10.1146/annurev.ph.52.030190.001353. [DOI] [PubMed] [Google Scholar]
  43. Törnquist K. Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells. Biochem Biophys Res Commun. 1991 Oct 31;180(2):860–866. doi: 10.1016/s0006-291x(05)81144-1. [DOI] [PubMed] [Google Scholar]
  44. Yue T. L., Stadel J. M., Sarau H. M., Friedman E., Gu J. L., Powers D. A., Gleason M. M., Feuerstein G., Wang H. Y. Platelet-activating factor stimulates phosphoinositide turnover in neurohybrid NCB-20 cells: involvement of pertussis toxin-sensitive guanine nucleotide-binding proteins and inhibition by protein kinase C. Mol Pharmacol. 1992 Feb;41(2):281–289. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES