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).

The ability of the lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) to promote the release of the organic osmolyte taurine in response to hypoosmotic stress has been examined. Incubation of SH-SY5Y neuroblastoma cells under hypoosmotic conditions (230 mOsM) resulted in a time-dependent release of taurine that was markedly enhanced (3-7-fold) by the addition of micromolar concentrations of either S1P or LPA. At optimal concentrations, the effects of S1P and LPA on taurine efflux were additive and mediated via distinct receptors. Inclusion of 1,9-dideoxyfoskolin, 5-nitro-2-(3-phenylpropylamino benzoic acid, or 4-[(2-butyl-6,7-dicloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]-butanoic acid blocked the ability of both lysophospholipids to enhance taurine release, indicating the mediation of a volume-sensitive organic osmolyte and anion channel. Both S1P and LPA elicited robust increases in intracellular calcium concentration that were attenuated by the removal of extracellular calcium, abolished by the depletion of intracellular calcium with thapsigargin, and were independent of phosphoinositide turnover. Taurine efflux mediated by S1P and LPA was unaffected by the removal of extracellular calcium but was attenuated by depletion of intracellular calcium (34 -38%) and by inhibition of protein kinase C (PKC) with chelerythrine (38 -72%). When intracellular calcium was depleted and PKC was inhibited, S1P-or LPAstimulated taurine efflux was inhibited by 80%. Pretreatment of the cells with pertussis toxin, toxin B, or cytochalasin D had no effect on lysophospholipid-stimulated taurine efflux. The results indicate that both S1P and LPA receptors facilitate osmolyte release via a phospholipase C-independent mechanism that requires the availability of intracellular calcium and PKC activity.

When retinal explants from goldfish are grown on a polycation substratum, a marked tendency for directionality of neurite outgrowth is observed. While the direct relevance to nerve growth in vivo is not known, the phenomenon is interpreted as reflecting an inherent helicity of the neurites.

The nature of the proteins synthesized in the goldfish retina and axonally transported to the tectum during optic nerve regeneration has been examined. Electrophoretic analysis of labeled soluble retinal proteins by fluorography verified our previous observation of a greatly enhanced synthesis of the microtubule subunits. In addition, labeling of a tubulin-like protein in the retinal particulate fraction was also increased during regeneration. Like soluble tubulin, the particulate material had an apparent MW of 53-55K and could be tyrosylated in the presence of cycloheximide and [3H]tyrosine. Comparison of post-crush and normal retinal proteins by two-dimensional gel electrophoresis also revealed a marked enhancement in the labeling of two acidic 68-70K proteins. Analysis of proteins slowly transported to the optic tectum revealed changes following nerve crush similar to those observed in the retina, with enhanced labeling of both soluble and particulate tubulin and of 68-70K polypeptides. the most striking change in the profile of rapidly transported protein was the appearance of a labeled 45k protein which was barely detectable in control fish.

The ability of muscarinic cholinergic receptors (mAChRs) to regulate the volume-sensitive efflux of two organic osmolytes, namely, taurine and D-aspartate, from human SH-SY5Y neuroblastoma cells has been examined. Incubation of the cells with hypoosmolar buffers resulted in an efflux of both osmolytes, with the threshold for release occurring at approximately 225 mOsM for taurine and D-aspartate. Inclusion of oxotremorine-M (Oxo-M), a muscarinic agonist, resulted in a marked enhancement of the volume-dependent efflux of both osmolytes and increased the threshold osmolarity for taurine and D-aspartate release to 340 (isotonic) and 320 mOsM, respectively. Maximum agonist stimulation of osmolyte release (350% of basal) was observed in the range of 225 to 250 mOsM. Oxo-M-stimulated osmolyte efflux was inhibited by muscarinic antagonists with a rank order of ,4]benzodiazepin-6-one, a pharmacological profile identical to that obtained for M 3 mAChRstimulated phosphoinositide hydrolysis. Agonist-stimulated efflux of both osmolytes could be inhibited by inclusion of either anion channel blockers known to inhibit the volume-sensitive organic anion channel (VSOAC) or by a tyrosine kinase inhibitor ␣-cyano-(3,4-dihydroxy)cinnamonitrile. The results indicate that the activation of M 3 mAChRs on SH-SY5Y neuroblastoma facilitates the ability of these cells to respond to very limited reductions in osmolarity via a release of osmolytes. mAChR-stimulated osmolyte efflux is mediated via a VSOAC and seems to require the activity of a tyrosine kinase.Although most cells possess homeostatic mechanisms for the maintenance of cell volume, these are particularly important to cells in the central nervous system (CNS) because of restrictions of the skull. Even modest alterations in brain volume can have profound effects on cell-cell signaling because the spatial relationship between neurons, astrocytes, and the extracellular space becomes compromised. Brain swelling, which can occur in response to conditions such as hyponatremia, inappropriate secretion of antidiuretic hormone, or after polydypsia, can lead to the compression of small blood vessels, and subsequently, cerebral anoxia and ischemia. Death can result from the displacement of brain parenchyma through the foramen magnum and the ensuing cardiac and respiratory arrest (Pasantes-Morales et al., 2000). To counter these deleterious changes, neural cells initially restore their osmotic balance via a loss of K ϩ and Cl Ϫ ions. However, because large changes in ion concentrations can adversely impact excitability, cells subsequently use "compatible" or nonperturbing organic osmolytes to counter changes in osmolarity without compromising cell function. In the CNS, the three quantitatively major organic osmolytes are taurine, glutamate, and myo-inositol. Organic osmolytes are released from neural cells via a volume-sensitive organic anion channel (VSOAC), a channel that has been extensively characterized both electrophysiologically and pharmacologically, although its molecular s...

Goldfish retinas were examined for changes in the labeling pattern of protein during regeneration of retinal ganglion cell axons following unilateral optic nerve crush. At various times after optic nerve crush the normal retinas were incubated in vitro with [3H] Following intra-orbital crush of the optic nerve, in goldfish, the retinal ganglion cell must grow out a new axon several millimeters long. This process not only implies a substantial increase in the rate of protein synthesis but may also involve a selective increase in the synthesis of the proteins necessary for the renewal of axoplasm and replacement of structural elements of the axon. Formation of cell-specific markers involved in retinotectal recognition may be increased~during this period (10). There might, in addition, be an initial decrease in the synthesis of components necessary to maintain the functional integrity of the nerve ending until the growing nerve reaches the optic tectum (11).This report presents comparisons of the in vitro protein labeling pattern of normal goldfish retina with that of retinas removed at various times after optic nerve crush (postcrush retina). A double-labeling technique coupled with sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (12) revealed an increased amino-acid incorporation into a protein fraction with properties of the microtubule subunit, tubulin. MATERIALS AND METHODSGoldfish (Carassius auratus), 6-7 cm in body length, were anesthetized with tricaine methane sulfonate prior to an intraorbital crush of the right optic nerve. The left optic nerve remained intact so that the left retina served as a control. Following surgery, the goldfish were stored in groups of 50 to 80 in 30 gallon tanks at 20-22°, and were fed daily.Fish were dark-adapted for 30 min prior to removal of retinas in order to facilitate separation of the retina from the pigment epithelium. After hemisection of the eye, the retina was floated free from the pigment layer with 0.86% saline and detached by cutting at the optic disk. The retinas were then rinsed and stored in ice-cold saline or incubation medium for up to 1 hr prior to incubation.In were evaporated to dryness under nitrogen immediately before use. After evaporation, each was resuspended in a small volume of incubation medium. The purity of each isotopic preparation was checked by paper chromatography. The incubation medium was that of Dunlop et al. (13): N-2-bydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 25 mM; MgSO4, 1.3 mM; CaCl2, 2.6 mM; K2HPO4, 1.2 mM; KCI, 5.9 mM; NaCl, 106.5 mM; glucose, 12 mM; and NaOH to pH 7.4.Retinas (in groups of 5 to 20) were pre-incubated for 2 min at 25°in 1.9 ml of incubation medium. Labeled precursor (0.1 ml) was then added (1.0-4.0 ,uCi per retina) and incubation was continued in air in a Dubnoff metabolic shaker at 250. All incubations using double-labeled methionine were carried out for 1 hr. The reaction was stopped by dilution with 2 volumes of ice-cold incubation medium containing 2 mM L-methionine. After c...

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 blockersresults 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. Keywords: calcium, muscarinic cholinergic receptors, myoinositol, osmolyte, protein kinase C, volume regulation. Regulation of cell volume is essential for many physiological processes and is of prime importance to the CNS, because of the restricted volume of the skull. Brain cells can swell in response to either changes in plasma osmolarity (hypoosmotic swelling) or from changes in intracellular ion and water distribution (isotonic swelling). The latter is also referred to as cellular or cytotoxic edema (Kimelberg 2000; Pasantes-Morales et al. 2000). Hypo-osmotic swelling frequently occurs as a result of hyponatremia, which is associated with clinical conditions such as congestive heart failure, nephrotic syndrome and hepatic cirrhosis. Water overload may also occur in some psychiatric disorders, such as schizophrenia, or in athletes and in instances of the inappropriate secretion of anti-diuretic hormone. The majority of symptoms observed are neurological and include disorientation, mental confusion and seizures.In response to hypo-osmotic stress, neural cells swell, and to restore osmotic balance a loss of K + and Cl -ions is initially observed. However, as large changes in ion concentrations can adversely impact cell excitability, cells subsequently utilize 'compatible' or 'non-perturbing' osmolytes which are specifically designed to counter changes in osmolarity without compromising cell function. Three distinct classes of osmolytes can be identified, namely (i) amino acids, such as glutamate or taurine, (ii) methylamines, such as betaine and glycerophosphorylcholine, and (iii) polyols, such...

The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-[3H]methylscopolamine [( 3H]NMS) and [3H]quinuclidinylbenzilate 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 microM), 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 116 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilycholine 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.