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. 1989 Aug;415:1–18. doi: 10.1113/jphysiol.1989.sp017708

Characteristics of a 5-hydroxytryptamine-sensitive adenylate cyclase in intact and intracellularly perfused squid axons.

T J Allen 1
PMCID: PMC1189163  PMID: 2561784

Abstract

1. Cyclic AMP metabolism was studied in intact and intracellularly perfused axons. 2. Cyclic AMP content of intact axons lay within the range 10-100 nmol kg-1 axoplasm. This was increased by exposure to caffeine (2-fold) and to 5-HT (15-fold). The caffeine-sensitive cyclic AMP increase was 30-fold larger in the presence of 5-HT. 3. A reduction in sodium concentration from the sea water bathing intact axons attenuated the 5-HT-evoked increase in cyclic AMP content, but had little effect on resting cyclic AMP. This effect was partially reversed by exclusion of external calcium, and suggests that free calcium plays a role in cyclic AMP homeostasis. 4. Prolonged exposure of intact axons to 5-HT (up to 3 h) led to apparent desensitization of the cyclic AMP response. 5. Intracellular perfusion can be used as a method to study adenylate cyclase in a single axon, simply by measuring the cyclic AMP content of the emerging perfusate. 6. Intracellular perfusion revealed micromolar requirements for internal GTP (K0.5 approximately 1-10 microM) and external 5-HT (K0.5 approximately 1-10 microM); a detailed investigation of this observation was limited due to the progressive loss of 5-HT-evoked adenylate cyclase activity with time. This slow loss was not seen during Gpp(NH)p (guanylylimidodiphosphate), NaF or forskolin activation of cyclase activity. 7. In perfused axons, an increase in intracellular calcium stimulated cyclase activated by 100 microM-forskolin but inhibited cyclase activated by 500 microM-Gpp(NH)p or 10 mM-NaF. A reduction in intracellular magnesium from 10 to 4-5 mM attenuated the effects of 5-HT-evoked cyclase activity. 8. Study of the perfused axon allows characterization of the intracellular requirements of a plasmalemmal transduction system which activates adenylate cyclase, whilst maintaining ionic asymmetry across the cell membrane.

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Selected References

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

  1. Adam-Vizi V., Allen T. J., Baker P. F. The effects of nitroprusside and putative agonists on guanylate cyclase activity in squid giant axons. Biochim Biophys Acta. 1988 Mar 3;938(3):461–468. doi: 10.1016/0005-2736(88)90144-7. [DOI] [PubMed] [Google Scholar]
  2. Allen T. J., Baker P. F. Comparison of the effects of potassium and membrane potential on the calcium-dependent sodium efflux in squid axons. J Physiol. 1986 Sep;378:53–76. doi: 10.1113/jphysiol.1986.sp016207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker P. F., Blaustein M. P., Hodgkin A. L., Steinhardt R. A. The influence of calcium on sodium efflux in squid axons. J Physiol. 1969 Feb;200(2):431–458. doi: 10.1113/jphysiol.1969.sp008702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baker P. F., Hodgkin A. L., Ridgway E. B. Depolarization and calcium entry in squid giant axons. J Physiol. 1971 Nov;218(3):709–755. doi: 10.1113/jphysiol.1971.sp009641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnbaumer L., Yang P. C. Studies on receptor-mediated activation of adenylyl cyclases. I. Preparation and description of general properties of an adenylyl cyclase system in beef renal medullary membranes sensitive to neurohypophyseal hormones. J Biol Chem. 1974 Dec 25;249(24):7848–7856. [PubMed] [Google Scholar]
  6. Brandt D. R., Ross E. M. Catecholamine-stimulated GTPase cycle. Multiple sites of regulation by beta-adrenergic receptor and Mg2+ studied in reconstituted receptor-Gs vesicles. J Biol Chem. 1986 Feb 5;261(4):1656–1664. [PubMed] [Google Scholar]
  7. Brandt D. R., Ross E. M. GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates. J Biol Chem. 1985 Jan 10;260(1):266–272. [PubMed] [Google Scholar]
  8. Brezina V., Eckert R., Erxleben C. Modulation of potassium conductances by an endogenous neuropeptide in neurones of Aplysia californica. J Physiol. 1987 Jan;382:267–290. doi: 10.1113/jphysiol.1987.sp016367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brostrom M. A., Brostrom C. O., Breckenridge B. M., Wolff D. J. Calcium-dependent regulation of brain adenylate cyclase. Adv Cyclic Nucleotide Res. 1978;9:85–99. [PubMed] [Google Scholar]
  10. Buxton I. L., Brunton L. L. Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem. 1983 Sep 10;258(17):10233–10239. [PubMed] [Google Scholar]
  11. Cassel D., Selinger Z. Mechanism of adenylate cyclase activation through the beta-adrenergic receptor: catecholamine-induced displacement of bound GDP by GTP. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4155–4159. doi: 10.1073/pnas.75.9.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cerione R. A., Staniszewski C., Caron M. G., Lefkowitz R. J., Codina J., Birnbaumer L. A role for Ni in the hormonal stimulation of adenylate cyclase. Nature. 1985 Nov 21;318(6043):293–295. doi: 10.1038/318293a0. [DOI] [PubMed] [Google Scholar]
  13. Clark R. B. Desensitization of hormonal stimuli coupled to regulation of cyclic AMP levels. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1986;20:151–209. [PubMed] [Google Scholar]
  14. Evans P. D., Reale V., Villegas J. The role of cyclic nucleotides in modulation of the membrane potential of the Schwann cell of squid giant nerve fibre. J Physiol. 1985 Jun;363:151–167. doi: 10.1113/jphysiol.1985.sp015701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ferguson K. M., Higashijima T., Smigel M. D., Gilman A. G. The influence of bound GDP on the kinetics of guanine nucleotide binding to G proteins. J Biol Chem. 1986 Jun 5;261(16):7393–7399. [PubMed] [Google Scholar]
  16. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  17. Iyengar R., Birnbaumer L. Hormone receptor modulates the regulatory component of adenylyl cyclase by reducing its requirement for Mg2+ and enhancing its extent of activation by guanine nucleotides. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5179–5183. doi: 10.1073/pnas.79.17.5179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lin M. C., Salomon Y., Rendell M., Rodbell M. The hepatic adenylate cyclase system. II. Substrate binding and utilization and the effects of magnesium ion and pH. J Biol Chem. 1975 Jun 10;250(11):4246–4252. [PubMed] [Google Scholar]
  19. Londos C., Salomon Y., Lin M. C., Harwood J. P., Schramm M., Wolff J., Rodbell M. 5'-Guanylylimidodiphosphate, a potent activator of adenylate cyclase systems in eukaryotic cells. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3087–3090. doi: 10.1073/pnas.71.8.3087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McArdle H., Mullaney I., Magee A., Unson C., Milligan G. GTP analogues cause release of the alpha subunit of the GTP binding protein, GO, from the plasma membrane of NG108-15 cells. Biochem Biophys Res Commun. 1988 Apr 15;152(1):243–251. doi: 10.1016/s0006-291x(88)80706-x. [DOI] [PubMed] [Google Scholar]
  21. Neer E. J., Clapham D. E. Roles of G protein subunits in transmembrane signalling. Nature. 1988 May 12;333(6169):129–134. doi: 10.1038/333129a0. [DOI] [PubMed] [Google Scholar]
  22. OIKAWA T., SPYROPOULOS C. S., TASAKI I., TEORELL T. Methods for perfusing the giant axon of Loligo pealii. Acta Physiol Scand. 1961 Jun;52:195–196. doi: 10.1111/j.1748-1716.1961.tb02218.x. [DOI] [PubMed] [Google Scholar]
  23. Pawlson L. G., Lovell-Smith C. J., Manganiello V. C., Vaughan M. Effects of epinephrine, adrenocorticotrophic hormone, and theophylline on adenosine 3', 5'-monophosphate phosphodiesterase activity in fat cells. Proc Natl Acad Sci U S A. 1974 May;71(5):1639–1642. doi: 10.1073/pnas.71.5.1639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rasenick M. M., Stein P. J., Bitensky M. W. The regulatory subunit of adenylate cyclase interacts with cytoskeletal components. Nature. 1981 Dec 10;294(5841):560–562. doi: 10.1038/294560a0. [DOI] [PubMed] [Google Scholar]
  25. Rodbell M., Lin M. C., Salomon Y., Londos C., Harwood J. P., Martin B. R., Rendell M., Berman M. Role of adenine and guanine nucleotides in the activity and response of adenylate cyclase systems to hormones: evidence for multisite transition states. Adv Cyclic Nucleotide Res. 1975;5:3–29. [PubMed] [Google Scholar]
  26. Rodbell M. The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature. 1980 Mar 6;284(5751):17–22. doi: 10.1038/284017a0. [DOI] [PubMed] [Google Scholar]
  27. Ross E. M., Gilman A. G. Biochemical properties of hormone-sensitive adenylate cyclase. Annu Rev Biochem. 1980;49:533–564. doi: 10.1146/annurev.bi.49.070180.002533. [DOI] [PubMed] [Google Scholar]
  28. Ross E. M., Howlett A. C., Ferguson K. M., Gilman A. G. Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J Biol Chem. 1978 Sep 25;253(18):6401–6412. [PubMed] [Google Scholar]
  29. Seamon K. B., Daly J. W. Forskolin: its biological and chemical properties. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1986;20:1–150. [PubMed] [Google Scholar]
  30. Shuster M. J., Camardo J. S., Siegelbaum S. A., Kandel E. R. Cyclic AMP-dependent protein kinase closes the serotonin-sensitive K+ channels of Aplysia sensory neurones in cell-free membrane patches. 1985 Jan 31-Feb 6Nature. 313(6001):392–395. doi: 10.1038/313392a0. [DOI] [PubMed] [Google Scholar]
  31. Sibley D. R., Lefkowitz R. J. Molecular mechanisms of receptor desensitization using the beta-adrenergic receptor-coupled adenylate cyclase system as a model. Nature. 1985 Sep 12;317(6033):124–129. doi: 10.1038/317124a0. [DOI] [PubMed] [Google Scholar]
  32. Smigel M. D. Purification of the catalyst of adenylate cyclase. J Biol Chem. 1986 Feb 5;261(4):1976–1982. [PubMed] [Google Scholar]
  33. Sternweis P. C., Gilman A. G. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4888–4891. doi: 10.1073/pnas.79.16.4888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Strada S. J., Martin M. W., Thompson W. J. General properties of multiple molecular forms of cyclic nucleotide phosphodiesterase in the nervous system. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;16:13–29. [PubMed] [Google Scholar]
  35. Strada S. J., Thompson W. J. Multiple forms of cyclic nucleotide phosphodiesterases: anomalies or biologic regulators? Adv Cyclic Nucleotide Res. 1978;9:265–283. [PubMed] [Google Scholar]
  36. VILLEGAS R., VILLEGAS G. M. Characterization of the membranes in the giant nerve fiber of the squid. J Gen Physiol. 1960 May;43:73–103. doi: 10.1085/jgp.43.5.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wells J. N., Hardman J. G. Cyclic nucleotide phosphodiesterases. Adv Cyclic Nucleotide Res. 1977;8:119–143. [PubMed] [Google Scholar]
  38. Westcott K. R., La Porte D. C., Storm D. R. Resolution of adenylate cyclase sensitive and insensitive to Ca2+ and calcium-dependent regulatory protein (CDR) by CDR-sepharose affinity chromatography. Proc Natl Acad Sci U S A. 1979 Jan;76(1):204–208. doi: 10.1073/pnas.76.1.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wolff D. J., Brostrom C. O. Properties and functions of the calcium-dependent regulator protein. Adv Cyclic Nucleotide Res. 1979;11:27–88. [PubMed] [Google Scholar]

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