Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1977 Dec 15;168(3):549–555. doi: 10.1042/bj1680549

A study of the kinetics of the muscarinic effect on phosphatidylinositol and phosphatidic acid metabolism in rat brain synaptosomes.

J C Miller
PMCID: PMC1183804  PMID: 606252

Abstract

The uptake of [32P]phosphate into phosphatidylinositol and phosphatidate was measured in synaptosomes incubated in Krebs-Ringer bicarbonate buffer, pH7.4. The apparent dissociation constants for acetylcholine and carbamoylcholine was estimated from the increase in 32P uptake caused by these agents. These apparent constants were similar for both phosphatidylinositol and phosphatidate and were 2.7 +/- 0.5 MICROmeter for acetylcholine and 12 +/- 2 micrometer for carbamoylcholine when Ca2+ concentration was 0.75 mM. Under the same conditions the inhibition of the carbamoylcholine-induced increase in 32P uptake, caused by atropine, is consistent with atropine being a competitive inhibitor, with an apparent inhibition constant of 0.35 +/- 0.05 micrometer. The apparent constants were dependent on the Ca2+ concentration, and were greater in 2.54 mM-Ca2+. The former values for the kinetic constants are similar to the muscarinic-receptor dissociation constant, which indicates that the binding of the agonist to the receptor may be rate-limiting in this series of reactions when the Ca2+ concentration is 0.75 mM.

Full text

PDF
549

Selected References

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

  1. Alberts P., Bartfai T. Muscarinic acetylcholine receptor from rat brain. Partial purification and characterization. J Biol Chem. 1976 Mar 25;251(6):1543–1547. [PubMed] [Google Scholar]
  2. BROSSARD M., QUASTEL J. H. Studies of the cationic, and acetylcholine, stimulation of phosphate incorporation into phospholipids in rat brain cortex in vitro. Can J Biochem Physiol. 1963 May;41:1243–1256. [PubMed] [Google Scholar]
  3. Birdsall N. J., Hulme E. C. Biochemical studies on muscarinic acetylcholine receptors. J Neurochem. 1976 Jul;27(1):7–16. doi: 10.1111/j.1471-4159.1976.tb01536.x. [DOI] [PubMed] [Google Scholar]
  4. Burgen A. S., Hiley C. R., Young J. M. The properties of muscarinic receptors in mammalian cerebral cortex. Br J Pharmacol. 1974 Jun;51(2):279–285. doi: 10.1111/j.1476-5381.1974.tb09658.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgen A. S., Spero L. The effects of calcium and magnesium on the response of intestine smooth muscle to drugs. Br J Pharmacol. 1970 Nov;40(3):492–500. doi: 10.1111/j.1476-5381.1970.tb10630.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DE ROBERTIS E., PELLEGRINO DE IRALDI A., RODRIGUEZ DE LORES GARNAIZ G., SALGANICOFF L. Cholinergic and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J Neurochem. 1962 Jan-Feb;9:23–35. doi: 10.1111/j.1471-4159.1962.tb07489.x. [DOI] [PubMed] [Google Scholar]
  7. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  8. Farrow J. T., O'Brien R. D. Binding of atropine and muscarone to rat brain fractions and its relation to the acetylcholine receptor. Mol Pharmacol. 1973 Jan;9(1):33–40. [PubMed] [Google Scholar]
  9. GARRY P. J., ROUTH J. I. A MICRO METHOD FOR SERUM CHOLINESTERASE. Clin Chem. 1965 Feb;11:91–96. [PubMed] [Google Scholar]
  10. GRAY E. G., WHITTAKER V. P. The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J Anat. 1962 Jan;96:79–88. [PMC free article] [PubMed] [Google Scholar]
  11. Hokin-Neaverson M. Acetylcholine causes a net decrease in phosphatidylinositol and a net increase in phosphatidic acid in mouse pancreas. Biochem Biophys Res Commun. 1974 Jun 4;58(3):763–768. doi: 10.1016/s0006-291x(74)80483-3. [DOI] [PubMed] [Google Scholar]
  12. Hokin L. E. Effects of calcium omission on acetylcholine-stimulated amylase secretion and phospholipid synthesis in pigeon pancreas slices. Biochim Biophys Acta. 1966 Jan 25;115(1):219–221. doi: 10.1016/0304-4165(66)90066-3. [DOI] [PubMed] [Google Scholar]
  13. Jafferji S. S., Michell R. H. Muscarinic cholinergic stimulation of phosphatidylinositol turnover in the longitudinal smooth muscle of guinea-pig ileum. Biochem J. 1976 Mar 15;154(3):653–657. doi: 10.1042/bj1540653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones L. M., Michell R. H. Breakdown of phosphatidylinositol provoked by muscarinic cholinergic stimulation of rat parotid-gland fragments. Biochem J. 1974 Sep;142(3):583–590. doi: 10.1042/bj1420583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LOLLEY R. N. THE CALCIUM CONTENT OF ISOLATED CEREBRAL TISSUES AND THEIR STEADY-STATE EXCHANGE OF CALCIUM. J Neurochem. 1963 Sep;10:665–676. doi: 10.1111/j.1471-4159.1963.tb08938.x. [DOI] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. Lapetina E. G., Brown W. E., Michell R. H. Muscarinic cholinergic stimulation of phosphatidylinositol turnover in isolated rat superior cervical sympathetic ganglia. J Neurochem. 1976 Mar;26(3):649–651. doi: 10.1111/j.1471-4159.1976.tb01530.x. [DOI] [PubMed] [Google Scholar]
  18. Lapetina E. G., Michell R. H. Effects of acetylcholine on incorporation of (14C)glucose into phosphatidylinositol and on phosphatidylinositol breakdown in subcellular fractions from cerebral cortex. J Neurochem. 1974 Jul;23(1):283–287. doi: 10.1111/j.1471-4159.1974.tb06949.x. [DOI] [PubMed] [Google Scholar]
  19. Michell R. H. Inositol phospholipids and cell surface receptor function. Biochim Biophys Acta. 1975 Mar 25;415(1):81–47. doi: 10.1016/0304-4157(75)90017-9. [DOI] [PubMed] [Google Scholar]
  20. Michell R. H., Jafferji S. S., Jones L. M. Receptor occupancy dose--response curve suggests that phosphatidyl-inositol breakdown may be intrinsic to the mechanism of the muscarinic cholinergic receptor. FEBS Lett. 1976 Oct 15;69(1):1–5. doi: 10.1016/0014-5793(76)80640-0. [DOI] [PubMed] [Google Scholar]
  21. Neudoerffer T. S., Lea C. H. Antioxidants for the (thin-layer) chromatography of lipids. J Chromatogr. 1966 Jan;21(1):138–140. doi: 10.1016/s0021-9673(01)91276-2. [DOI] [PubMed] [Google Scholar]
  22. Oron Y., Löwe M., Selinger Z. Incorporation of inorganic [32P] phosphate into rat parotid phosphatidylinositol. Induction through activation of alpha adrenergic and cholinergic receptors and relation to K+ release. Mol Pharmacol. 1975 Jan;11(1):79–86. [PubMed] [Google Scholar]
  23. Pumphrey A. M. Incorporation of [32P]orthophosphate into brain-slice phospholipids and their precursors. Effects of electrical stimulation. Biochem J. 1969 Mar;112(1):61–70. doi: 10.1042/bj1120061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. REDMAN C. M., HOKIN L. E. STIMULATION OF THE METABOLISM OF PHOSPHATIDYLINOSITOL AND PHOSPHATIDIC ACID IN BRAIN CYTOPLASMIC FRACTIONS BY LOW CONCENTRATIONS OF CHOLINERGIC AGENTS. J Neurochem. 1964 Mar;11:155–163. doi: 10.1111/j.1471-4159.1964.tb06126.x. [DOI] [PubMed] [Google Scholar]
  25. Richards C. D., Sercombe R. Calcium, magnesium and the electrical activity of guinea-pig olfactory coex in vitro. J Physiol. 1970 Dec;211(3):571–584. doi: 10.1113/jphysiol.1970.sp009294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roscoe H. G., Goldstein R., Riccardi B. A., Fahrenbach M. J. Lipid biosynthesis in normal and lipid infiltrated rabbit irises. Arch Biochem Biophys. 1970 May;138(1):329–337. doi: 10.1016/0003-9861(70)90314-0. [DOI] [PubMed] [Google Scholar]
  27. SKIPSKI V. P., PETERSON R. F., SANDERS J., BARCLAY M. THIN-LAYER CHROMATOGRAPHY OF PHOSPHOLIPIDS USING SILICA GEL WITHOUT CALCIUM SULFATE BINDER. J Lipid Res. 1963 Apr;4:227–228. [PubMed] [Google Scholar]
  28. Schacht J., Agranoff B. W. Effects of acetylcholine on labeling of phosphatidate and phosphoinositides by ( 32 P) orthophosphate in nerve ending fractions of guinea pig cortex. J Biol Chem. 1972 Feb 10;247(3):771–777. [PubMed] [Google Scholar]
  29. Schacht J., Agranoff B. W. Stimulation of hydrolysis of phosphatidic acid by cholinergic agents in guinea pig synaptosomes. J Biol Chem. 1974 Mar 10;249(5):1551–1557. [PubMed] [Google Scholar]
  30. Trifaró J. M. The effect of Ca++ omission on the secretion of catecholamines and the incorporation of orthophosphate-32P into nucleotides and phospholipids of bovine adrenal medulla during acetylcholine stimulation. Mol Pharmacol. 1969 Jul;5(4):424–427. [PubMed] [Google Scholar]
  31. Yagihara Y., Bleasdale J. E., Hawthorne J. N. Effects of acetylcholine on the incorporation of (32P)orthophosphate in vitro into the phospholipids of subsynaptosomal, membranes from guinea-pig brain. J Neurochem. 1973 Jul;21(1):173–190. doi: 10.1111/j.1471-4159.1973.tb04237.x. [DOI] [PubMed] [Google Scholar]
  32. Yamamura H. I., Snyder S. H. Muscarinic cholinergic binding in rat brain. Proc Natl Acad Sci U S A. 1974 May;71(5):1725–1729. doi: 10.1073/pnas.71.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES