Stephen K. Fisher

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 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).

A Pleckstrin Homology Domain Specific for Phosphatidylinositol 4,5-Bisphosphate (PtdIns-4,5-P2) and Fused to Green Fluorescent Protein Identifies Plasma Membrane PtdIns-4,5-P2 as Being Important in Exocytosis

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca2+. One of these priming steps involves the maintenance of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P2is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P2 important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P2 that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4,5-P2 and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P2, showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca2+ entry and suggest that plasma membrane PtdIns-4,5-P2 is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P2 in some manner alters calcium channel function in chromaffin cells.

Enhanced Coupling of Neonatal Muscarinic Receptors in Rat Brain to Phosphoinositide Turnover

Abstract: The relationship between the density of the muscarinic receptor in developing rat cerebral cortex and its coupling to phosphoinositide turnover is examined. Tissue slices from rats of various ages were incubated with myo‐[2‐3H]inositol, and the effect of carbamoylcholine on the release of total inositol phosphates was determined. Binding of [3H]quinuclidinyl benzilate was determined in the same tissue. Although muscarinic receptor density in day‐18 embryonic cortex was only 5% of that in the adult, the maximal response of stimulated phosphoinositide turnover to carbamoylcholine (1–10 mM) was at the adult level (i.e., threefold increase). Comparison of the dependence of the turnover on carbamoylcholine concentration revealed that in neonates, the dose‐response curve was shifted to the left, giving a half‐maximal effect at concentrations approximately tenfold lower than that in the adult. In addition, the partial muscarinic agonists oxotremorine‐2 and bethanechol were both more efficacious in young rats than in adults. The differences could not be accounted for either by alterations in agonist affinity for the receptor or by the presence of “spare” muscarinic receptors. These results indicate that muscarinic receptors in fetal and newborn rat cerebral cortex are more efficiently coupled to stimulation of phosphoinositide turnover than in the adult.

Renewed interest in the polyphosphoinositides

Abstract The significance of the enhanced cellular phosphatidate and phosphatidylinositol turnover which occurs in response to specific extracellular messengers has been the subject of much interest and speculation. Until recently, much less attention has been paid to the presence of two quantitatively minor phosphorylated derivatives of phosphatidylinositol, known collectively as the polyphosphoinositides. These lipids have an extremely rapid 32 P turnover rate and are presumed to be localized predominantly in the plasma membranes. Their turnover now appears to be linked with that of phosphatidate and phosphatidylinositol, and is discussed here in relation to the consequences of ligand-receptor interactions.

Calcium and the muscarinic synaptosomal phospholipid labeling effect.

Abstract: The role of calcium in the muscarinic phospholipid labeling effect in synaptosomes has been investigated. In the absence of added calcium, acetylcholine doubled phosphatidate labeling and increased phosphatidy linositol labeling 40% in synaptosomes when incubated in a medium that contained [32P]orthophosphate. Inclusion of calcium or the omission of magnesium resulted in a marked elevation of acetylcholine‐stimulated phosphatidylinositol labeling (70–80%) while phosphatidate stimulation was unaltered. Calciumchelating agents, EGTA and EDTA, reduced the stimulated labeling of both phosphatidate and phosphatidylinositol, but this inhibition could be reversed by calcium addition. The calcium ionophore A23187, which promotes entry of calcium into cells, selectively increased labeling of both phosphatidate and phosphatidyl‐inositol. This effect, unlike acetylcholine‐stimulated labeling, was not blocked by the addition of atropine. The calcium dependency of the acetylcholine stimulation, on the one hand, and the insensitivity of the ionophore to a muscarinic antagonist, on the other, argue strongly that the acetylcholine‐receptor interaction regulates calcium mobilization and that the latter is linked to the stimulated labeling of phosphatidate and phosphatidylinositol.

A Putative M3 Muscarinic Cholinergic Receptor of High Molecular Weight Couples to Phosphoinositide Hydrolysis in Human SK-N-SH Neuroblastoma Cells

The M1‐selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N‐[3H]methylscopolamine ([3H]NMS) and [3H]qui‐nuclidinylbenzilate from intact human SK‐N‐SH neuroblastoma cells with a low affinity (Ki= 869–1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF‐DX 116 {11–2[[2‐[(diethylamino)methyl]‐1‐piperidinyl]‐acetyl]‐5,11‐dihydro‐6H‐pyrido[2,3‐b][1,4]benzodiazepin‐6‐one} bound to the mAChRs with a very low affinity (Ki= 6.0 μM), 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki= 7.2 nM). 4‐DAMP was also 1,000‐fold more effective than AF‐DX 1 16 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilylcholine mustard) suggest that the size of the SK‐N‐SH mAChR (Mr= 81.000–98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr=66,000), an M1‐enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.

Perinatal hypoxic-ischemic brain injury enhances quisqualic acid-stimulated phosphoinositide turnover.

Abstract: In an experimental model of perinatal hypoxic‐ischemic brain injury, we examined quisqualic acid (Quis)‐stimulated phosphoinositide (PPI) turnover in hippocampus and striatum. To produce a unilateral forebrain lesion in 7‐day‐old rat pups, the right carotid artery was ligated and animals were then exposed to moderate hypoxia (8% oxygen) for 2.5 h. Pups were killed 24 h later and Quis‐stimulated PPI turnover was assayed in tissue slices obtained from hippocampus and striatum, target regions for hypoxic‐ischemic injury. The glutamate agonist Quis (10‐4M) preferentially stimulated PPI hydrolysis in injured brain. In hippocampal slices of tissue derived from the right cerebral hemisphere, the addition of Quis stimulated accumulation of inositol phosphates by more than ninefold (1,053 ± 237% of basal, mean ± SEM, n = 9). In contrast, the addition of Quis stimulated accumulation of inositol phosphates by about fivefold in the contralateral hemisphere (588 ± 134%) and by about sixfold in controls (631 ± 177%, p < 0.005, comparison of ischemic tissue with control). In striatal tissue, the corresponding values were 801 ± 157%, 474 ± 89%, and 506 ± 115% (p < 0.05). In contrast, stimulation of PPI turnover elicited by the cho‐linergic agonist carbamoylcholine, (10‐4 or 10‐2M) was unaffected by hypoxia‐ischemia. The results suggest that prior exposure to hypoxia‐ischemia enhances coupling of excitatory amino acid receptors to phospholipase C activity. This activation may contribute to the pathogenesis of irreversible brain injury and/or to mechanisms of recovery.

Differentiated Human NT2‐N Neurons Possess a High Intracellular Content of myo‐Inositol

Abstract: myo‐Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2‐N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2‐N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2‐N cells, respectively. The high concentrations of inositol in NT2‐N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2‐N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2‐N neurons vs 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.

Activation of muscarinic cholinergic receptors enhances the volume‐sensitive efflux of myo‐inositol from SH‐SY5Y neuroblastoma cells

A mechanism used by cells to regulate their volume under hypo‐osmotic conditions is the release of organic osmolytes, one of which is myo‐inositol. The possibility that activation of phospholipase‐C‐linked receptors can regulate this process has been examined for SH‐SY5Y neuroblastoma cells. Incubation of cells with hypo‐osmolar buffers (160–250 mOsm) led to a biphasic release of inositol which persisted for up to 4 h and could be inhibited by inclusion of anion channel blockers – results which indicate the involvement of a volume‐sensitive organic anion channel. Inclusion of oxotremorine‐M, a muscarinic cholinergic agonist, resulted in a marked increase (80–100%) in inositol efflux under hypo‐osmotic, but not isotonic, conditions. This enhanced release, which was observed under all conditions of hypo‐osmolarity tested, could be prevented by inclusion of atropine. Incubation of the cells with either the calcium ionophore, ionomycin, or the phorbol ester, phorbol 12‐myristate 13‐acetate, partially mimicked the stimulatory effect of muscarinic receptor activation when added singly, and fully when added together. The ability of oxotremorine‐M to facilitate inositol release was inhibited by removal of extracellular calcium, depletion of intracellular calcium or down‐regulation of protein kinase C. These results indicate that activation of muscarinic cholinergic receptors can regulate osmolyte release in this cell line.