Rueben A. Gonzales

Cholinergic‐ and Adrenergic‐Stimulated Inositide Hydrolysis in Brain: Interaction, Regional Distribution, and Coupling Mechanisms

Abstract: Carbachol and norepinephrine were used as agonists to compare and contrast cholinergic and adrenergic stimulation of inositide breakdown in rat brain slices. Carbachol acts through a muscarinic (possibly M1) receptor and norepinephrine acts through an α1 adrenoceptor. Studies in cerebral cortical slices indicated that both agonists stimulated the production of inositol‐1‐ phosphate and glycerophosphoinositol. Although the initial rates for the stimulation of inositol phosphate release were similar for the two ligands, the response to norepinephrine continued for 60 min and was larger compared with carbachol which plateaued at 30 min. The presence of carbachol did not affect the ED50 for norepinephrine. Concentrations of carbachol near the ED50 in combination with norepinephrine resulted in an additive response whereas maximal concentrations of carbachol and norepinephrine resulted in a less than additive response in the cortex. This negative interaction was also seen in the hippocampus and hypothalamus but not in the striatum, brainstem, spinal cord, olfactory bulb, or cerebellum. Norepinephrine had a larger response than carbachol in the hippocampus, striatum, and spinal cord, but the reverse was true in the olfactory bulb. Manganese (1 mM) stimulated the incorporation of [3H]inositol into phosphatidylinositol (PtdIns) four‐ to fivefold but not into polyphosphoinositides. The stimulation by manganese of PtDIns labelling increased the monstimulated release of inositol phosphates but did not affect the stimulated release of inositol phosphates by carbachol or norepineph rine. These data suggest that: (a) there may be a common pool of inositides that can be hydrolyzed by cholinergic and/or adrenergic agonists in certain brain areas; (b) manganese may stimulate the labelling of a pool of PtdIns that does not interact with the receptor‐coupled pool of inositides; and (c) there may be differences in the basic mechanisms coupling receptor occupation and inositide breakdown for cholinergic and adrenergic receptors.

Receptor-mediated inositide hydrolysis is a neuronal response: comparison of primary neuronal and glial cultures.

Cholinergic and adrenergic receptor-stimulated inositide hydrolysis was studied in neuronal and glial cells cultured from brains of 1-day-old Wistar-Kyoto rats. Incubation of the cells with [3H]inositol led to the incorporation of radioactivity specifically into inositol phospholipids. Labeling of the membrane lipids reached a maximum in 2-3 days. Receptor-stimulated breakdown of inositides was determined by following the accumulation of inositol phosphates after incubation of the labeled cells for 60 min with carbachol or norepinephrine in the presence of 10 mM lithium. Carbachol (1 mM) stimulated inositol phosphate production in neurons 30 times higher than that seen in glia. The response stimulated by norepinephrine (75 microM) was 6 times higher in neurons than glia. The response to carbachol was blocked by atropine, and the norepinephrine-induced response was inhibited by prazosin suggesting that the receptors mediating the responses were muscarinic and alpha 1-adrenergic, respectively. These results suggest that muscarinic cholinergic and alpha 1-adrenergic stimulated inositide hydrolysis is primarily a neuronal response and that this biochemical event may be important for transmembrane signaling which occurs during neurotransmission.

Phorbol esters inhibit agonist-stimulated phosphoinositide hydrolysis in neuronal primary cultures

The effects of phorbol esters on neurotransmitter-stimulated phosphoinositide (PI) hydrolysis in neurons in primary culture were investigated. Ten-day-old neuronal cultures were incubated with [3H]inositol for 2-3 days, exposed to phorbol esters, and the release of [3H]inositol phosphates was measured in the presence of 10 mM lithium. Pretreatment of the neuronal cultures with 1 microM phorbol myristate acetate (PMA) inhibited alpha 1, muscarinic, and glutamate receptor-mediated PI hydrolysis in a time-dependent manner with maximal inhibition observed after a 20-30 min preincubation. The active beta-phorbol didecanoate inhibited stimulated PI hydrolysis, but its stereo-isomer alpha-phorbol didecanoate was without effect at 1 microM. PMA was about 10 times more potent at inhibiting PI hydrolysis stimulated by norepinephrine and glutamate compared to carbachol. The order of potency of the various phorbol esters for inhibition of stimulated PI hydrolysis and the differences between active and inactive stereoisomers suggests that the activation of protein kinase C may mediate the inhibitory effects. Thus, stimulation of neuronal protein kinase C may represent a mechanism for the regulation of agonist-stimulated PI hydrolysis.

Receptors for phorbol esters are primarily localized in neurons: Comparison of neuronal and glial cultures

Binding of [3H]PDB has been measured in the present study to determine the levels of protein kinase C in the neuronal and astrocytic glial cells in culture from rat brain. Binding of [3H]PDB to homogenates of cultured neuronal cells from the brains of normotensive and hypertensive rats was time-dependent and specific. The relative potency for competition by various phorbol esters to [3H]PDB binding was TPA > β-PDD > POE > α-PDD ≥4αphorbol. Scatchard analysis showed that neuronal cultures from normotensive rat brains contained 2–3 fold more phorbol ester receptors compared with the glial cultures from the same brains. No differences in theKd andBmax were observed between neuronal cultures from normotensive and spontaneously hypertensive rat brains. These studies suggest that the phorbol ester receptors are primarily localized in neuronal cells.

Variations in Membrane Sensitivity of Brain Region Synaptosomes to the Effects of Ethanol In Vitro and Chronic In Vivo Treatment

Abstract: : The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer‐344 rats either were fed a nutritionally complete ethanol‐containing liquid diet for 5 months or pair‐fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30–500 mM) decreased the polarization values in synaptosomes from sucrose‐control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater than that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol‐fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanols membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.

Effect of ethanol and aging on histamine release and membranes of mast cells

The effects of ethanol on histamine release from mast cells were compared to ethanols effects on membrane order of mast cell membranes and synaptosomes in young (6 month) and old (24 month) Fischer 344 rats. Concanavalin A (con A) stimulated histamine release in a concentration dependent manner. Ethanol (10-500 mM) inhibited con A stimulated release while having no effect on nonstimulated release in both young and old rats. Ethanols effect on membrane order of synaptosomes and mast cell plasma membranes was estimated by measuring the fluorescence polarization of diphenylhexatriene. Ethanol (10-500 mM) decreased the polarization of synaptosomes to the same degree in young and old rats. The polarization of mast cell membranes was also decreased by ethanol but to a greater degree than synaptosomes. The ethanol induced changes in polarization correlated (r2 = 0.99) with ethanols inhibition of con A stimulated histamine release from mast cells. These findings suggest that mast cells may be more sensitive to membrane disordering by ethanol than synaptosomes. In addition, we have demonstrated that mast cells may be a useful model system for studying ethanol effects on stimulus-secretion coupling. No differences were found between rats 6 and 24 months for histamine release (with or without ethanol) or membrane order of mast cells or synaptosomes.

The Effects of Ethanol on Receptor Activated Phospholipid Cascades

Ethanol is one of the most widely used drugs in the world. The resulting alcohol abuse and alcoholism is a major health and social problem. An understanding of the actions of ethanol are fundamental to a rational approach to treating alcoholism and to reversing the pathology associated with excessive ethanol consumption.