Adenylate Cyclase of <i>Torpedo</i> Synaptosomes Is Inhibited by Calcium and Not Affected by Muscarinic Ligands

Pinchasi, Irit; Michaelson, Daniel M.

doi:10.1111/j.1471-4159.1982.tb07894.x

Cited by 7 publications

(5 citation statements)

References 38 publications

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“…We were also unable to confirm clearly biochemical localization data from Pinchasi and Michaelson (1982) who reported adenylate cyclase activity to be associated with synaptosomal membranes. The reason may lie in low residual enzyme activity and unspecific deposits we had to contend with in many experiments.…”

Section: Discussioncontrasting

confidence: 71%

“…U1-trastructural preservation was found to be best when 50 mM cacodylate-nitrate buffer at pH 7.4 with 200 mM sucrose was used, though 50 mM Tris-acetate was postulated for biochemical measurements in the electric organ by Pinchasi and Michaelson (1982) and though in many histochemical media 50 mM Tris-maleate buffer was used (Reik et al 1970;Howell and Whitefield 1972;Dini and del Rosso 1983). Pipes buffer which we used routinely in synaptosomal preparations for electron microscopy (Miiller et al 1983) did not satisfactorily maintain structural integrity.…”

Section: Discussionmentioning

confidence: 96%

“…Its role is basically known as an initiator of the enzymatic phosphorylation of certain proteins via activation of protein kinases. This phenomenon is also of relevance for transmission of the chemical signal across the synaptic cleft and -as shown by Pinchasi and Michaelson (1982) for Torpedo synaptosomes on the presynaptic side of the endplate. In their studies, biochemical data on conditions for maximal enzyme activity revealed adenylate cyclase (EC 4.6.1.1) to be activated by 0.05 mM guanosine triphosphate (GTP).…”

Section: Introductionmentioning

confidence: 92%

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Ultrahistochemical localization of adenylate cyclase activity in the electric organ ofTorpedo marmorata

1985

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The lead pyrophosphate precipitation technique was used to visualize adenylate cyclase activity with the electron microscope in unfixed electric organ and synaptosomes of Torpedo marmorata, with special attention to presynaptic membranes. Specificity of the deposition of reaction product was ensured by using 5'-adenylyl imidodiphosphate as substrate and 5'-guanylyl imidodiphosphate and sodium fluoride as activators. Under suitable conditions a reaction product was deposited on the Schwann cell, on presynaptic vesicles, on the inner side of membranes of cisternae and on glycogen granules of the presynaptic region of the endplate. In some cases, a precipitate was also found on postsynaptic membranes of the synaptic cleft and on mitochondria. In isolated synaptosomes localization of the reaction product was identical with that of minced tissue. However, most strikingly, on presynaptic membranes no precipitate was ever found, neither in pieces of electric organ nor in isolated synaptosomes. Furthermore, the extended membrane system of the postsynaptic region of the electroplax remained always free of lead pyrophosphate precipitate.

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Section: Discussioncontrasting

confidence: 71%

Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 92%

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Ultrahistochemical localization of adenylate cyclase activity in the electric organ ofTorpedo marmorata

1985

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“…In Torpedo synaptosome preparations, muscarinic agonist binding is regulated by GTP, but the mAChR is not coupled to inhibition of adenylate cyclase activity (Pinchesi & Michaelson, 1982), suggesting a role for guanine nucleotide regulation other than for coupling to adenylate cyclase. A variety of reports examining the physiological effects of pertussis toxin also suggest that N[ may have functions not directly related to alterations of cAMP levels (Nakamura & Ui, 1983;Cronin et al, 1983;Dorflinger & Schonbrunn, 1983;De Wildt et al, 1983).…”

Section: Development Of the Chronotropic Effect Of Muscarinicmentioning

confidence: 94%

Ontogenesis of physiological responsiveness and guanine nucleotide sensitivity of cardiac muscarinic receptors during chick embryonic development

Halvorsen¹,

Nathanson²

1984

Biochemistry

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Atria isolated from 4-day chick embryos were much less responsive to the negative chronotropic effect of muscarinic agonists than were atria from 5- or 8-day embryos, even though the density of muscarinic acetylcholine receptors (mAChR) was similar at all these ages. The mAChR in hearts from 4-day embryos were also significantly less susceptible to regulation of receptor number by in vivo agonist treatment and required a 2-5-fold greater dose of the muscarinic agonist carbachol to achieve a decrease in receptor number equivalent to that observed in 5- or 8-day embryonic hearts. When 4-day atrial membranes were assayed in physiological buffers, agonist binding to the mAChR was not regulated by GTP unless a sulfhydryl reducing agent was present. Receptors from 5- and 8-day embryos did not require addition of a sulfhydryl reducing agent in order to see guanine nucleotide effects on agonist binding. Even in the presence of a sulfhydryl reducing agent, carbachol binding to the mAChR in 4-day membranes was much less sensitive to guanyl-5'-yl imidodiphosphate (GppNHp) than binding to mAChR in 5- or 8-day membranes. In addition, forskolin-activated adenylate cyclase activity was much less sensitive to inhibition by GppNHp in membranes from 4-day atria than from 5- and 8-day atria. The GTP-binding component (NI) which couples the mAChR to inhibition of adenylate cyclase activity was examined by covalent modification with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…In other systems (Watanabe et al, 1978;Lichtshtein et al, 1979;Jakobs et al, 1979), a guanine nucleotide effect has been linked to muscarinic receptor inhibition of adenylate cyclase. However, a recent study using Torpedo synaptosomes suggests that whilst adenylate cyclase activity is activated by guanine nucleotides, it is not affected by muscarinic ligands (Pinchasi and Michaelson, 1982). The organization of the muscarinic pathway in Torpedo may, thus, be different from that in other tissues which have been studied and this may reflect differences between pre-and postsynaptic receptors.…”

Section: Discussionmentioning

confidence: 94%

Muscarinic Acetylcholine Receptors in Torpedo Electric Organ: Effect of Guanine Nucleotides

Dowdall

Strange

Golds

1983

Journal of Neurochemistry

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The effect of guanine nucleotides on the binding properties of presynaptic muscarinic receptors has been studied in a membrane preparation from the electric organ of Torpedo marmorata by measuring the competitive displacement of the radiolabelled antagonist, [3H]quinuclidinyl benzilate, by nonradioactive muscarinic ligands. The binding of the antagonists, atropine, scopolamine and pirenzepine was to a single class of sites [slope factors (pseudo Hill coefficients) close to 1] and was unaffected by 0.1 mM GTP. The binding of the N-methylated antagonists, N-methylatropine and N-methyl-scopolamine was more complex (slope factors less than 1) but also insensitive (N-methylatropine) to 0.1 mM GTP. Agonist binding was complex and could be resolved into two binding sites with relatively high and low affinities. The proportion of high-affinity sites varied with the nature of the agonist (15-80%). Agonist binding was depressed by 0.1 mM GTP, and the order of sensitivity was oxotremorine-M greater than carbamoylcholine greater than muscarine greater than acetylcholine greater than arecoline greater than oxotremorine. The binding of pilocarpine, a partial agonist, was unaffected by GTP. With carbamoylcholine as a test ligand the GTP effect on agonist binding was half-maximal at 12 microM. GDP and guanylylimidodiphosphate produced comparable inhibition of carbamoylcholine binding, but GMP and cyclic GMP were ineffective, as were various adenine nucleotides. Analysis of agonist binding in terms of a two-site model indicates that the predominant effect of guanine nucleotides is to reduce the number of sites of higher affinity.

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Adenylate Cyclase of Torpedo Synaptosomes Is Inhibited by Calcium and Not Affected by Muscarinic Ligands

Cited by 7 publications

References 38 publications

Ultrahistochemical localization of adenylate cyclase activity in the electric organ ofTorpedo marmorata

Ultrahistochemical localization of adenylate cyclase activity in the electric organ ofTorpedo marmorata

Ontogenesis of physiological responsiveness and guanine nucleotide sensitivity of cardiac muscarinic receptors during chick embryonic development

Muscarinic Acetylcholine Receptors in Torpedo Electric Organ: Effect of Guanine Nucleotides

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