Irit Pinchasi

On the interaction of drugs with the cholinergic nervous system--V. Characterization of some effects induced by physostigmine in mice: in vivo and in vitro studies.

Abstract Four systemic effects induced by s.c. injection of physosthmine and neostigmine to mice were investigated by the “quadro-test” procedure, a novel design in which salivation, tremor, hypothermia and the rotarod-effects are simultaneously and continuously recorded. The time profiles of these effects were used to construct dose-response curves, which were compared to those of brain acetylcholinesterase (AcChE) inhibition by the drug. A good relationship was found between the dose and time dependency of these pharmacological and biochemical parameters, as well as between the relative potencies of physostigmine and neostigmine and their affinity to the enzyme. Using scopolamine. Hbr and its quaternary analogue, as well as tertiary cholinergic agonists and other cholinesterase agents, it was found that the physostigmine-induced hypothermia is mostly a central-muscarinic response, while the tremor is most probably a non-muscarinic peripheral effect. The peripheral-muscarinic origin of the salivation was confirmed. The effects measured in the rotarod test were found to be muscarinic and mixed central-peripheral for physostigmine, and non-muscarinic for neostigmine. The lethality caused by physostigmine seems to be partially centrally mediated, while that of neostigmine is peripheral.

On the interaction of drugs with the cholinergic nervous system—III: Tolerance to phencyclidine derivatives: In vivo and in vitro studies

Abstract A possible contribution of metabolic processes to the tolerance to cyclohexamine (l-(l-phenylcyclohexyl) ethylamine) was investigated by determining the kinetics of brain and liver uptake of the labeled drug. A similar time course was found for both naive and tolerant mice. In addition, the amount of the drug uptaken by the brain was found to be linearly dependent on the dose injected in both groups. The possibility of adaptive changes in brain enzymes was investigated using mouse brain acetylcholinesterase (AcChE, E.C.3.1.1.7) as a model of a putative enzyme, affected by phencyclidine derivatives. Although brain AcChE is believed to be chronically affected by these drugs in vivo , no measurable changes could be observed in the amount, the affinity towards diverse ligands or the kinetic properties of this enzyme, between naive, cyclohexamine-tolerant and physostigmine tolerant mice. Possible changes in receptors as the mechanism of tolerance induction were tested by determining the amount of the central muscarinic receptor and its affinity towards a highly specific antimuscarinic ligand in vitro . When comparing naive animals to mice tolerant to cyclohexamine, physostigmine and oxotremorine, no measurable differences could be found in any of these parameters. Repeated injections of cyclohexamine together with scopolamine prevented tolerance development to the former. The possibility of homeostatic events as tolerance mechanism is presented and discussed.

On the interaction of drugs with the cholinergic nervous system--IV. Tolerance to oxotremorine in mice: in vivo and in vitro studies.

Abstract A new procedure was used to follow continuously and simultaneously four systemic effects induced by oxotremorine in mice: salivation, tremor, hypothermia and those measured in the rotarod test. Using an equipotent dose of methoxotremorine, it was found that apart from salivation, the other systemic effects are centrally originated. By comparing the complete dose-response curves for these systemic effects in naive and tolerant mice, it was found that: (1) Salivation is the most sensitive effect in naive mice; (2) All curves shift in parallel to the right on continuous exposure to oxotremorine, in a manner that seems to be dose-dependent; (3) the tolerance to oxotremorine is reversible. Since oxotremorine-tolerant mice were found to be cross-tolerant to various tertiary anti-cholinesterase agents and cholinergic agonists, the involvement of the muscarinic receptor in the tolerance to oxotremorine was investigated, using two different approaches. (1) Continuous blockade of the receptor by scopolamine in vivo prevented tolerance development to oxotremorine; this effect was found to depend on the time of scopolamine administration relative to oxotremorine injection, and on the systemic effect measured. (2) The amount of receptor and its affinity towards a specific ligand were determined in vitro ; no significant differences were found between naive and oxotremorine-tolerant animals. The significance of these results in the elucidation of possible tolerance mechanisms is discussed.

On the interaction of drugs with the cholinergic nervous system

Tolerance to physostigmine salicylate was induced in mice using various schedules of s.c. injections. The rate and degree of tolerance development were assessed by comparing the ED50 values (equipotent doses) and by comparing the peak effects induced by a constant dose. These were measured on four systemic responses induced by the drug — hypothermia, tremor, salivation, and the effects measured in the rotarod test. The degree of tolerance development was found to be dose-dependent with a maximal achievable tolerance for every dose. The tolerance development to the four systemic effects differed in time course: tolerance to the hypothermia was induced even with daily injections, while tolerance to the salivation and rotarod effects could be detected only when the drug was given every 4 h. No tolerance developed to the tremor with any of the schedules and doses used. The maximal achievable tolerance degree and the pattern of changes of the duration were different for each systemic effect. The tolerance was found to be reversible, with different rates of recovery, for the different effects. The tolerance state could not be correlated with changes in the pattern of brain acetylcholinesterase (AcChE) inhibition by physostigmine in vivo or with changes in the rate of the enzymes spontaneous reactivation.Scopolamine. HBr given 10 min before physostigmine prevented tolerance development. In addition, cross-tolerance to various muscarinic agonists and cholinesterase inhibitors was found in the physostigmine-tolerant mice. The correlation between these results and our previous findings concerning possible biochemical adaptations is presented and discussed.

Newly Synthesized and Preformed Acetylcholine Are Released from Torpedo Synaptosomes by Different Pathways

Abstract: In this study, we investigated the mechanisms underlying the release of preformed and of newly synthesized acetylcholine (ACh) from isolated Torpedo nerve terminals (synaptosomes). This was pursued by examining and comparing the effects of anticytoskeletal and anticalmodulin drugs and of activating the presynaptic muscarinic ACh receptors on the release of preformed endogenous ACh and of newly synthesized radiolabeled ACh. The anticytoskeletal drugs vinblastine, cytochalasin B, and colchicine inhibit the Ca2+‐dependent K+mediated release of newly synthesized radiolabeled ACh, but have no effect on the release of preformed ACh. By contrast, the muscarinic agonist oxotremorine markedly inhibits the release of preformed ACh, but has little effect on the release of newly formed ACh. Treatment of the synaptosomes with the calmodulin antagonist trifluoperazine inhibits the release of both ACh pools concomitantly. These findings show that preformed and newly synthesized ACh are released by different routes and suggest that their secretion is mediated by converging pathways. The significance of these results in view of the previously demonstrated preferential release of newly synthesized ACh is discussed.

Differential inhibition of the release of endogenous and newly synthesized acetylcholine from Torpedo synaptosomes by presynaptic muscarinic receptors

Activation of Torpedo presynaptic muscarinic acetylcholine (ACh) receptors with the agonist oxotremorine (20 μM) results in the inhibition of Ca2+‐dependent release of endogenous ACh from Torpedo synaptosomes. This effect is reversed by the muscarinic antagonist atropine (1 μM) which, by itself, has no effect. In contrast, under the same conditions the amount of newly synthesized radiolabeled [3H]ACh released is not affected by muscarinic ligands. These findings suggest that presynaptic muscarinic inhibition in the Torpedo is due to interference with the mobilization of ACh from a storage pool.

Cholinergic nerve terminals contain ascorbic acid which induces Ca2+‐dependent release of acetylcholine and ATP from isolated Torpedo synaptic vesicles

We have shown that a soluble, low-molecular weight, alkali-labile and oxidation-sensitive factor, prepared from the electric organ of Torpedo ocellata, induces Cap-dependent acetylcholine (ACh) release from isolated Torpedo synaptic vesicles [ 1,2]. Diverse activities of ascorbic acid in the brain have been reported [3-81. These include effects on metabolism of biogenic amines [9] and on release of neurotransmitters from isolated mammalian vesicles [lo]. We therefore tested the possibility that ascorbic acid may be the soluble factor present in Torpedo and thus a naturally-occurring mediator of ACh release in vivo.

Factors required for calcium dependent acetylcholine release from isolated torpedo synaptic vesicles

Abstract The release of acetylcholine (Ach) from Torpedo synaptic vesicles has been investigated. Factors have been found which induce Ca+2 dependent Ach release from the synaptic vesicles. In the absence of these factors, the vesicles are not affected by Ca+2. Addition of a soluble factor to the vesicles induces a Ca+2-dependent release of their Ach. This secretion is enhanced by a non-vesicular membranous component which, by itself, does not induce the Ca+2-dependent release. These results demonstrate that vesicular Ach release may be studied in vitro and thus will enable the study, at the molecular level, of the biochemical events underlying neurotransmission.

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

Abstract: Cholinergic synaptosomes isolated from the electric organ of Torpedo contain membrane‐bound adenylate cyclase activity (∼6 pmol/mg proteidmin), which is dependent on the presence of guanine nucleotides. The activity is strongly dependent on temperature and only slightly affected by NaCl. The Torpedo adenylate cyclase is completely inhibited by low levels of free Ca2+ (K0∼ 0.5 μM). This effect is not altered by either trifluoperazine or addition of exogenous calmodulin. Ca3+ has no effect on the activation step of the adenylate cyclase by guanyl‐5′‐yl imidodiphosphate (GppNHp), and Mn2+ abolishes the Ca2+‐dependent inhibition of cyclic AMP synthesis. These findings suggest that Ca2+ exerts its effect by direct interaction with a site located on the catalytic subunit. Torpedo synaptosomes contain presynaptic inhibitory muscarinic receptors. The binding of muscarinic agonists to the receptors is modulated (to lower affinity) by GTP. However, muscarinic ligands, examined under a variety of assay conditions, have no effect on adenylate cyclase activity. These results suggest that although both the muscarinic receptor and the adenylate cyclase are coupled to G proteins, they either interact with different G proteins or are situated in different regions of the presynaptic membrane.