Moshe Hershkowitz

The Modulation of Protein Phosphorylation and Receptor Binding in Synaptic Membranes by Changes in Lipid Fluidity: Implications for Ageing

Publisher Summary Phosphorylation and dephosphorylation of membrane proteins are intermediate steps in signal transduction by hormones and neurotransmitters. These processes are regulated by cyclic nucleotides and by ions, and are probably dependent on membrane dynamics. Complex, sequential membranal events can be classified into two categories: active (metabolically driven) and passive. Active processes are characterized by energy consumption, (for example, ATP-linked) which can be blocked by metabolic poisons and low temperatures. These processes are long-term in nature and require a specific compartmental strucutre. Most of these processes are associated with the cytoskeletal network. Passive processes, on the other hand, are directly related to diffusion (lateral, rotational or vertical) and are largely determined by membrane lipid fluidity. These processes do not require metabolic energy and can proceed in isolated membranes. Therefore, alteration of membrane lipid fluidity can, passively and instantaneously, modulate receptors, antigens and enzymes. This chapter describes the passive modulation of protein phosphorylation and receptor binding by in vitro or in vivo lipid manipulations.

Effect of cannabinoids on neurotransmitter uptake, atpase activity and morphology of mouse brain synaptosomes.

Δ1-Tetrahydrocannabinol (Δ1-THC) and cannabidiol (CBD), a psychoactive and a nonpsychoactive constituent of marijuana respectively, inhibit the uptake of 3H-labelled norepinephrine (NE), dopamine (DA), γ-aminobutyric acid (GABA) and serotonin (5-HT), by mouse brain synaptosomes. CBD is more effective than Δ1-THC in the inhibition of neurotransmitter uptake. At 5 × 10−5 M both CBD and Δ1-THC inhibit uptake by 60–100%. The one exception to the above is the differential effect of Δ1-THC and CBD on 5-HT uptake. At 10−6 M of Δ1-THC the uptake is twice that of the control value and at 5 × 10−5 M uptake is still equal to control value. At the former concentration CBD has no effect on 5-HT uptake whereas at the latter concentration a 50 per cent inhibition is observed. Both Δ1-THC and CBD inhibit Na+-K+-ATPase and Mg-ATPase activities; at 5 × 10−5 M inhibition amounts to 40 per cent. Electron microscopy reveals that synaptosomal preparations are highly damaged at 5 × 10−5 M. Thus inhibition of uptake could stem from either failure of ATPase activity, from disruption of synaptosomes, or from both.

Influence of calcium on phosphorylation of a synaptosomal protein

Synaptosomal proteins isolated from rat cerebral cortex were phosphorylated endogeneously in the presence of [gamma-32P]ATP. The phosphorylated proteins were found to be membrane bound by differential and density gradient centrifugation. In contrast to the phosphorylation of all synaptosomal proteins, phosphorylation of one protein (C), 41 000--43 000 daltons, was inhibited by Mg2+ and stimulated by Ca2+. In addition, the ionophores X537A and A23187, as well as papaverine, selectively enhanced phosphorylation of protein C without affecting phosphorylation of the outer proteins. Cyclic AMP did not influence the phosphorylation of protein C but markedly affected the phosphorylation of other synaptosomal proteins. It appears that the phosphorylation of protein C is stimulated by agents which trigger the release of neurotransmitters (Ca2+, X537A, A23187 and papaverine), and is inhibited by Mg2+, which inhibits release. It is proposed that the phosphorylation of protein C is related to membranal events underlying the release of neurotransmitters.

The retention of learning during metamorphosis of the crested newt (Triturus cristatus)

Abstract Larvae of the urodeles, Triturus cristatus , can be taught a simple discrimination task for food reward. The preference established by training is retained by the adult newts after metamorphosis. No significant change in the natural preference of these animals occurs after metamorphosis. Adult animals appear to be more difficult to train than the larvae.

The effects of dibutyryl cyclic AMP, theophylline and papaverine on the release of 3H-catecholamines from rat brain striatal and cortical synaptosomes.

Papaverine enhances the spontaneous release of 3H-dopamine from rat brain striatal synaptosomes and 3H-noradrenaline from cortical synaptosomes in a dose-dependent way. Neither dibutyryl cyclic AMP nor theophylline had any significant effect on either spontaneous or on potassium-evoked 3H-catecholamine release. It is suggested that the effect of papaverine on catecholamine release is not due to its phosphodiesterase inhibitory potency.

The effect of ACTH on rat brain synaptic plasma membrane lipid fluidity

The effect of ACTH on the lipid fluidity was examined in synaptic plasma membranes from rat forebrain. ACTH1-24 increased the fluidity of the synaptic plasma membranes in a dose-dependent way, the lowest effective dose being 10(-5) M. The shorter N-terminal fragment ACTH1-10 was not effective. The significance of this finding is discussed in relation to the known effects of ACTH on synaptic membrane phosphorylation.

The effect of in vivo treatment with (-) delta 1-tetrahydrocannabinol, and other psychoactive drugs on the in vitro uptake of biogenic amines.

Summary Since cannabinoids inhibit the uptake of biogenic amines into synaptosomes in vitro, the effects of chronic and acute (—)Δ1-tetrahydrocannabinol [(—)Δ1-THC] administration on the uptake of 3H-dopamine (3H-DA) into cortical and striatal synaptosomes from mouse brain were tested in the present study. To test whether the effects of (—)Δ1-THC on 3H-DA uptake are specific, (—)Δ1-THC was compared to (+)Δ6-THC administration. The uptake of 3H-DA into cortical synaptosomes increased in mice pretreated acutely with (—)Δ1-THC or cannabidiol (CBD) but not in mice pretreated with (+)Δ6-THC. The 3H-DA uptake was no longer increased after chronic (1 week) treatment with (—)Δ1-THC. The effect of in vivo treatment with (—)Δ1-THC on the in vitro uptake was not specific to 3H-DA, but enhanced also the uptake of 3H-norepinephrine (3H-NE), 3H-5-hydroxytryptamine (3H-5HT), and 3H-γ-aminobutyric acid (3H-GABA) into cortical or whole brain synaptosomes. Intravenous administration of dopamine or norepinephrine receptor antagonists, i.e. chlorpromazine, haloperidol or propanolol, enhanced the uptake of 3H-DA, and 3H-NE respectively into cortical synaptosomes. Pretreatment with uptake inhibitors such as the tricyclic antidepressants, amphetamine, or with the monoamine oxidase inhibitor, pargyline, caused an inhibition of the uptake of 3H-DA or 3H-NE into the isolated synaptosomes. It is suggested that in vivo (—)Δ1-THC does not act on uptake mechanism but on receptors.

Rapid modification of amphibian behavior by punishment

Specimens of Pleurodeles waltl which were kept in deep water and shocked whenever they ascended to the surface for air soon ascended less frequently than yoked controls which were shocked independently of their own behavior whenever the experimental animals were shocked. The results suggest that, under suitable conditions, amphibians are capable of rapid learning.

Effect of appetitive training on brain lysine level and incorporation into nuclear proteins

The content of free lysine in the brains of mice increased significantly during an appetitive training in which the mice were trained to touch a bar in order to get sweetened milk. The free lysine level reached a maximum at 20–30 min of training, and returned to control levels at 60 min. The specific activity of free lysine was significantly lower in the brains of trained mice than in controls at 20 and 30 min after either subcutaneous or intracerebral administration of the isotopically labeled compound. Subcutaneously injected radioactive lysine disappeared more rapidly from the blood of trained mice than from the blood of control mice during the interval from 20 to 60 min after injection. The specific activities of brain nuclear proteins from trained mice were significantly greater than those of controls after 20 min or more of training. These protein differences were more marked when expressed as relative specific activities that were corrected for changes of specific activity of free lysine that occurred during training.

The influence of different behavioral situations on the incorporation of [5-3H]orotic acid into RNA in the newtsTriturus cristatus andpleurodeles waltl

Summary The effects of two different training procedures on the incorporation of [5- 3H ]- orotic acid into total brain and liver RNA of the newts Triturus cristatus and Pleurodeles waltl are reported. Triturus cristatus trained to spatial discrimination reinforced by food or trained to avoid darkness by aversive electrical shocks incorporated more [5- 3 H]orotic acid into their brain RNA after training than did yoked newts or untreated newts. Pleurodeles waltl trained to avoid darkness incorporated more than the untreated group after training ceased while the yoked animals incorporated less than the untreated group into their brain RNA. No changes occurred in the incorporation into the liver RNA of any of the experimental newts.