Stefan R. Nahorski

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: I. Receptor Characterisation

Abstract: Characterisation of receptor‐mediated breakdown of inositol phospholipids in rat cortical slices has been performed using a direct assay which involves prelabelling with [3H]inositol. When slices were preincubated with [3H]inositol, lithium was found to greatly amplify the capacity of receptor agonists such as carbachol, noradrenaline, and 5‐hydroxytryptamine to increase the amount of radioactivity appearing in the inositol phosphates. Using a large variety of agonists and antagonists it could be shown that cholinergic muscarinic, α1,‐adrenoceptor, and histamine H1, receptors appear to be linked to inositol phospholipid breakdown in cortex. The large responses produced by receptor agonists allowed a clear discrimination between full and partial agonists as well as quantitative analysis of competitive antagonists for each receptor. Whereas carbachol and acetylcholine (in the presence of a cholinesterase inhibitor) were full agonists, oxotremorine and arecoline were only partial agonists. Very low concentrations of atropine shifted the carbachol dose‐response curve to the right and allowed inhibition constants for the antagonist to be easily calculated. The nicotinic antagonist, mecamylamine, was ineffective. Noradrenaline and adrenaline were full agonists at α1‐adrenoceptors, but phenylephrine and probably methoxamine were partial agonists. Prazosin, but not yohimbine, potently and competitively antagonised the noradrenaline inositol phospholipid response. Mepyramine but not cimetidine competitively antagonised the histamine response. These data provide strong confirmation for the potentiating effect of lithium on neurotransmitter inositol phospholipid breakdown and emphasise the ease with which functional responses at a number of cortical receptors can be characterised.

Lithium and the phosphoinositide cycle: an example of uncompetitive inhibition and its pharmacological consequences.

The ability of lithium to exert profound and selective psychopharmacological effects to ameliorate manic-depressive psychosis has been the focus of considerable research effort. There is increasing evidence that lithium exerts its therapeutic action by interfering with polyphosphoinositide metabolism in brain and prevention of inositol recycling by an uncompetitive inhibition of inositol monophosphatase. Stefan Nahorski, Ian Ragan and John Challiss discuss this unusual stimulus-dependent form of enzyme inhibition, emphasizing that the selectivity exhibited by lithium depends upon the degree of inositol lipid hydrolysis and polyphosphoinositide dephosphorylation.

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: II. Calcium Requirement

Abstract: The calcium requirement for agonist‐dependent breakdown of phosphatidylinositol and polyphos‐phoinositides has been examined in rat cerebral cortex. The omission of added Ca2+ from the incubation medium abolished [3H]inositol phosphate accumulation from prelabelled phospholipid induced by histamine, reduced that due to noradrenaline and 5‐hydroxytryptamine, but did not affect carbachol‐stimulated breakdown. EC50 values for agonists were unaltered in the absence of Ca2+. Removal of Ca2+ by preincubation with EGTA (0.5 mM) abolished all responses, but complete restoration was achieved by replacement of Ca2+. The EC50for Ca2+ for histamine‐stimulated [3H]inositol phosphate accumulation was 80 μM. Noradrenaline‐stimulated breakdown was antagonised by manganese (IC50 1.7 mM), but not by the calcium channel blockers nitrendipine or nimodipine (30 μM). The calcium ionophore A23187 stimulated phosphatidylinositol/polyphosphoinositide hydrolysis with an EC50 of 2 μM, and this response was blocked by EGTA. Omission of Ca2+ or preincubation with EGTA or Mn2+ (EC50= 230 μM) greatly enhanced the incorporation of [3H]inositol into phospholipids. The IC50for Ca2+ in inhibiting incorporation was 25 μM. The results show that different receptors mediating phosphatidylinositol/ polyphosphoinositide breakdown in rat cortex have quantitatively different Ca2+ requirements, and it is suggested that rigid opinions regarding phosphatidylinositol/ polyphosphoinositide breakdown as either cause or effect of calcium mobilisation in rat cortex are inappropriate.

[3H]rauwolscine and [3H]yohimbine binding to rat cerebral and human platelet membranes: Possible heterogeneity of α2-adrenoceptors

The specific binding of the alpha 2-adrenoceptor antagonists [3H]yohimbine and [3H]rauwolscine to membranes prepared from rat cerebral cortex and human platelets was rapid, reversible, saturable and of high affinity. Both ligands labelled an identical population of sites in cerebral or platelet membranes though the affinities of both agents were significantly lower in brain. The specific binding of [3H]yohimbine and [3H]rauwolscine in both tissues was displaced by various adrenergic agents in a manner that suggests that the labelled sites probably represent the alpha 2-adrenoceptor. There were, however, significant differences in the affinities of certain alpha-antagonists between cerebral and platelet preparations, even though these agents generated displacement curves with slopes close to unity in both tissues. Thus, whereas yohimbine, rauwolscine, WB4101 and corynanthine were all weaker in cerebral cortex, prazosin was significantly more potent at cortical than in platelet membranes. A possible heterogeneity of alpha 2-adrenoceptors is discussed.

Molecular recognition of inositol polyphosphates by intracellular receptors and metabolic enzymes

The discovery that inositol lipids are fundamentally involved in cell signalling has been one of the most significant recent advances in cell biology. In particular, there is now evidence that certain products of polyphosphoinositide metabolism play second messenger roles in most cells. Inositol 1,4,5-trisphosphate and perhaps inositol 1,3,4,5-tetrakisphosphate bind to specific receptors and regulate Ca2+ release from, and movement between, intracellular stores. The synthesis of novel analogues of these second messengers is now providing clues to the structural requirements at such receptors as well as for molecules with stability towards metabolic enzymes. Stefan Nahorski and Barry Potter discuss these developments with a view to future pharmacological intervention at these sites.

Differences between the release of radiolabelled and endogenous dopamine from superfused rat brain slices: Effects of depolarizing stimuli, amphetamine and synthesis inhibition

Direct comparisons between radiolabelled and endogenous dopamine (DA) release from superfused rat brain slices have been made. Striatal slices were prelabelled with [3H]dopamine ([3H]DA), then superfused at 0.5 ml/min and the released catecholamines analyzed by HPLC with electrochemical detection and the radioactivity present in superfusate fractions also counted. Two successive 50 mM K+ pulses released similar amounts of endogenous DA from striatal slices, but the second pulse released 50% less [3H]DA than the first. A K+ gradient (5-53 mM) released relatively more [3H]DA compared to endogenous DA at lower K+ than at higher K+ concentrations. Blockade of DA synthesis in vitro by 50 microM a-methyl-p-tyrosine greatly reduced K+-induced endogenous DA release without any major effect on [3H]DA release. Amphetamine (10 microM) greatly increased both basal DA release and release induced by a 5 microM veratrine pulse, but its effects were 3-4 times greater on endogenous than on [3H]DA release. Although a-methyl-p-tyrosine reduced both basal and veratrine-stimulated endogenous DA release from non-prelabelled tissue by over 50% in either the presence or absence of amphetamine, it did not decrease endogenous DA release from prelabelled tissue. These studies indicate that labelled and endogenous amine release do not always occur in parallel, and that major causes of discrepancy between them may include the presence of a large newly-synthesized component in endogenous release and the uneven distribution of labelled amine within endogenous releasable pools. The results also suggest that the prelabelling process itself may alter the pools contributing to subsequent endogenous release. In the light of these studies, the assumption that labelled amine release provides an accurate marker for endogenous release should be reconsidered.

Muscarinic receptor binding characteristics of a human neuroblastoma SK-N-SH and its clones SH-SY5Y and SH-EP1

The present study examines the muscarinic receptor binding characteristics of parent human neuroblastoma (SK-N-SH) and its neuroblast (SH-SY5Y) and epithelial-like (SH-EP1) clones using [3H]methylscopolamine [( 3H]NMS). Specific [3H]NMS binding to intact SK-N-SH and SH-SY5Y cells was saturable with a Kd of 0.2 nM and Bmax of 100-150 fmol/mg protein. Specific [3H]NMS binding to whole cell preparations of SH-EP 1 could not be detected. Pharmacological analysis of the binding site both in whole cells and membranes of SK-N-SH are indicative of an homogeneous receptor population possessing low affinity for the M1-selective antagonist pirenzepine. The muscarinic receptors expressed by the neuroblast clone, SH-SY5Y were further characterized and shown to have the properties of an homogeneous M3 subtype with low affinity for the M1-selective antagonist pirenzepine and the M2-cardioselective AFDX-116 but high affinity for 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP). In conclusion the SH-SY5Y neuroblastoma should provide an important human neuronal cell model with which to define the regulation of post-receptor events driven by a single receptor population.

Intracellular signalling: Receptor-specific messenger oscillations

The cytosolic molecule inositol-1,4,5-trisphosphate (InsP3) acts as a messenger to link receptors at the cell surface with alterations in calcium concentration inside the cell, but it is not clear how production of InsP3 is related to the often-complex calcium response. Here we use a fluorescent biosensor to visualize InsP3 synthesis in individual cells in real time and show that this is periodically switched on and off in a receptor-specific manner. Our findings are consistent with intracellular calcium oscillations being generated by either fluctuating or sustained concentrations of InsP3, which may allow diversity of signalling through the same signal-transduction pathway.

Inositol polyphosphates and neuronal calcium homeostasis.

Abstract Inositol phospholipids play a crucial role in translating many hormonal and neurotransmitter signals at cell surface receptors into appropriate cellular responses. Activation of a phosphoinositidase C generates two intracellular second messengers, inositol (1,4,5) trisphosphate [(1,4,5)IP 3 ] and diacylglycerol (DAG), that control a variety of cellular functions by the ability of the former to mobilize Ca 2+ from intracellular stores and the latter to activate protein kinase(s) C. Recent work suggests added complexity to this signalling system, perhaps indicative of further functional roles for inositol polyphosphates. These current ideas are discussed with some emphasis on the nature and possible mechanism of intracellular receptors for (1,4,5)IP 3 and (1,3,4,5)-tetrakisphosphate [(1,3,4,5)IP 4 ]. Potential effects on neuronal Ca 2+ homeostasis and consequent modification of membrane K + currents may play an important role in altering neuronal excitability to other incoming signals.

Dihydropyridine calcium channel activators and antagonists influence depolarization-evoked inositol phospholipid hydrolysis in brain

Increased inositol phospholipid hydrolysis induced by elevated extracellular K+ was directly monitored by assaying [3H]inositol phosphate accumulation following prelabelling of cerebral cortical slices with [3H]inositol. Depolarization evoked by K+ increased [3H]inositol phosphate accumulation with a 2-3-fold stimulation observed at 18 mM K+. Higher concentrations of K+ failed to further increase accumulation though a suppression of the incorporation of [3H]inositol into phospholipid at higher K+ could complicate these results. Slices incubated with the dihydropyridine calcium channel activator BAY-K-8644 resulted in a much increased response to 12 mM and 18 mM K+ with substantially smaller enhancement of basal (6 mM) or much higher (30 and 55 mM) K+. The [3H]inositol phosphate response induced by 18 mM K+ + 1 microM BAY-K-8644 was markedly reduced when incubations were performed in the presence of reduced Ca2+. Similarly, preincubation of slices with the dihydropyridine antagonist PN-200-110 suppressed the response to K+ and to K+ + BAY-K-8644. This effect was stereospecific with the (+)-enantiomer being at least 100-fold more potent than the (-)-enantiomer. These data provide primary evidence for functional dihydropyridine-sensitive calcium channels in brain.