Bernard W. Agranoff

Receptor Activation and Inositol Lipid Hydrolysis in Neural Tissues

Recent advances in our knowledge of the biochemistry, pharmacology, and cell biology of ligand-induced, stimulated turnover of inositol lipids in such diverse tissues as insect salivary glands, platelets, lymphocytes, and a number of exocrine glands have greatly altered our understanding of intracellular events leading to secretion, contraction, chemotaxis, and other cellular responses. Much of what has been learned regarding ligand-stimulated turnover of inosito1 lipids has come from nonneural systems, so that the presumption that signal transduction via this mechanism mediates significant steps in neural function must still be considered inferential. This reservation has been a major theme of a recent comprehensive review (Hawthorne, 1986). Nevertheless, that the brain contains large amounts of the substrates and enzymes of inositol lipid turnover and its associated second messenger systems and, further, that it is enriched with receptors that are linked to stimulated inositol lipid turnover argue strongly that the phosphoinositides indeed play an important role in brain function. There exist several general reviews on stimulated inositol lipid turnover (Bemidge, 1984; Bemdge and Irvine, 1984; Nishizuka, 1984, 1986; Hokin, 1985; Abdel-Latif, 1986; Downes, 1986; Williamson, 1986), including several that emphasize the nervous system (Downes, 1982, 1983; Fisher and Agranoff, 1986; Hawthorne, 1986; Nahorski et al., 1986). The present contribution emphasizes developments of neurochemical relevance since a review on the subject in this journal 8 years ago (Hawthorne and Pickard, 1979).

Inositol and higher inositol phosphates in neural tissues: homeostasis, metabolism and functional significance

Inositol phospholipids and inositol phosphates mediate well‐established functions in signal transduction and in Ca2+ homeostasis in the CNS and non‐neural tissues. More recently, there has been renewed interest in other roles that both myo‐inositol and its highly phosphorylated forms may play in neural function. We review evidence that myo‐inositol serves as a clinically relevant osmolyte in the CNS, and that its hexakisphosphate and pyrophosphorylated derivatives may play roles in such diverse cellular functions as DNA repair, nuclear RNA export and synaptic membrane trafficking.

Inositol Lipids and Signal Transduction in the Nervous System: An Update

Abstract: The role that inositol lipids play in cellular signaling events in eukaryotic cells remains one of the most intensively investigated areas of cell biology. In this respect, phosphoinositide‐mediated signal transduction in the CNS is no exception; major advances have been made since a previous review on this subject (Fisher and Agranoff, 1987). Not only have stimulated phosphoinositide turnover and its physiological sequelae been demonstrated repeatedly in a variety of neural preparations, but, in addition, the detailed molecular mechanisms underlying these events continue to unfold. Here we review the progress that has occurred in selected aspects of this topic since 1987. In the first two sections of this article, emphasis is placed on novel functional roles for the inositol lipids and on recent insights into the molecular characteristics and regulation of three key components of the phosphoinositide signal transduction system, namely, the inositol lipid kinases, phospholipases C (PLCs), and the inositol 1,4,5‐trisphosphate[I(1,4,5)P3] receptor. The metabolic fate of I(1,4,5)P3 in neural tissues, as well as its control, is also detailed. Later we focus on identification of the multiple receptor subtypes that are coupled to inositol lipid turnover and discuss possible strategies for intervention into phosphoinositide‐mediated signal transduction. Due to space limitations, an extensive evaluation of the diacylglycerol/protein kinase C (DAG/PKC) limb of the signal transduction pathway is not included (for reviews, see Nishizuka, 1988; Kanoh et al., 1990).

Chemical studies on memory fixation in goldfish.

Summary Puromycin and acetoxycycloheximide, antibiotics known to block selectively protein synthesis, also block the formation of memory of shock avoidance in the goldfish.

Puromycin Effect on Memory Fixation in the Goldfish

Puromycin injected intracranially into the goldfish produces impairment of memory for a shock-avoidance response. Intracranial injection of puromycin aminonucleoside, or of saline has no effect. Puromycin does not afject performance in naive or overtrained goldfish.

RAPID TRANSPORT OF PROTEIN IN THE OPTIC SYSTEM OF THE GOLDFISH

Abstract— Several amino acids, particularly [3H]proline and [3H]asparagine specifically and efficiently labelled rapidly transported proteins in the goldfish optic nerve and tectum after intraocular injection. Studies with these amino acids showed that the rapidly transported proteins moved as a discrete band at a rate which was temperature‐dependent, and was equal to 70‐100 mm per day at 20°C. Transported protein in the optic tectum was 80 per cent particulate and was found in synaptosomal, mitochondrial, and myelin fractions, but not in purified nuclei or ribosomes.

Muscarinic receptors in chromaffin cell cultures mediate enhanced phospholipid labeling but not catecholamine secretion.

Abstract: The addition of either carbachol or muscarinic agonists to cultured bovine adrenal chromaffin cells results in a selective stimulation of phosphatidate (PhA) and phosphatidylinositol (PhI) labeling from 32Pi and [3H]glycerol that can be inhibited by the inclusion of atropine, but not d‐tubocurarine. In contrast, increased catecholamine secretion is observed on the addition of carbachol or nicotinic agonists and is inhibited by d‐tubocurarine but not by atropine. Added calcium is essential for catecholamine secretion but not for stimulated phospholipid labeling. Chelation of endogenous Ca2+ with EGTA does, however, inhibit the stimulated phospholipid labeling. These results suggest that stimulated phospholipid labeling in the bovine chromaffin cell and catecholamine secretion are separate and distinct processes.

Actinomycin D Blocks Formation of Memory of Shock-Avoidance in Goldfish

When 2 micrograms of antinomycin D was injected intracranially into goldfish immediately after a training session, the formation of long-term memory of a shock-avoidance was blocked. The results are discussed in relation to similar findings with acetoxycycloheximide and puromycin in the goldfish and with apparently conflicting results in the mouse.

Explant culture of adult goldfish retina: A model for the study of CNS regeneration

Conditions are described for culture of retinal explants of adult goldfish which favour outgrowth of neuritic processes onto a substratum. A growth index to quantitate the outgrowth was developed. If the optic nerve is crushed several days prior to explantation, a marked enhancement of neuritic outgrowth is seen relative to control retinas. Histological examination of the explants revealed that retinal ganglion cells in explants from unoperated eyes became hypertrophied in vitro with a time course similar to that observed in vivo following optic nerve crush. Experiments with hemiaxotomized retinas indicate that the perikaryal regenerative response is mediated intracellularly.

COMPARISON OF THE FATTY ACIDS OF LIPIDS OF SUBCELLULAR BRAIN FRACTIONS

Abstract— Rat brain grey and white matter were fractionated to yield myelin, nerve terminal, synaptic vesicle, nerve terminal ‘ghost’, and microsomal fractions of white and grey matter. Ester‐type glycolipids were found in all fractions except myelin, while cerebrosides occurred in significant concentrations only in myelin and white microsomes. Comparison of the fatty acid profile of the ethanolamine‐ and serine‐containing phospholipids showed marked differences between myelin and the particles from grey matter, while the microsomes of white matter were of intermediate composition. Docosahexaenoic acid, a minor acid in myelin, was a major fatty acid in microsomes of grey and white matter. The fatty acid composition of sphingomyelin was distinctly different in the fractions derived from grey and white matter, clustering about stearate and nervonate in the latter, but only about stearate in the grey. Marked differences in the positional distribution of fatty acids were seen within phosphatidyl choline from myelin and nerve terminals. Ribonucleic acid was found in nerve terminal and synaptic vesicle fractions. The sphingosine found in the ganglioside from microsomes of both grey and white matter was similar with respect to distribution of the C18 and C20 homologues.