Multiple actions of fluoride ions upon the phosphoinositide cycle in the rat brain.

The effects of sodium fluoride upon basal and agonist-stimulated inositol phospholipid breakdown have been investigated in rat brain miniprisms. NaF concentration dependently increased basal inositol phospholipid breakdown, with a maximum effect being seen at 20 mM. NaF reduced the inositol phospholipid breakdown responses to stimulation by carbachol, noradrenaline, serotonin and quisqualate, but not to the stimulation produced by raising the assay [K+] from 6 to 18 mM. More detailed study demonstrated NaF to have a 'levelling' effect, reducing all InsP/(Lipid + InsP) values greater than 0.15 (i.e. produced by carbachol at raised [K+], noradrenaline and by 50 mM K+) to about this value. Time-course experiments indicated that NaF treatment reduced the rate of carbachol-stimulated inositol phospholipid breakdown up to this InsP/(Lipid + InsP) level and thereafter blocked further breakdown. Inhibitory effects upon carbachol-stimulated inositol phospholipid breakdown were not seen with forskolin, sodium nitroprusside or 8BrcGMP. Under conditions where there is no de novo synthesis of phosphoinositides from [3H]myo-inositol, NaF reduced the total Lipid + InsP labelling by about 20%. NaF in addition inhibits the activity of Ins(1,4)P2-phosphatase in cerebral cortical homogenates. It is concluded that fluoride ions inhibit agonist-stimulated inositol phospholipid breakdown via actions not only on G-proteins but also on phosphoinositide-specific phospholipase C substrate availability.

Effect of monovalent ions upon G proteins coupling muscarinic receptors to phosphoinositide hydrolysis in the rat cerebral cortex.

It has been suggested that K+, Li+ and Fl- affect the function of G proteins coupled to signal transducing enzymes. Lithium, at concentrations which were found to reduce forskolin-stimulated adenylate cyclase activity, was without effect on either membrane [3H]phosphatidylinositol-4,5-bisphosphate ([3H]PIP2) hydrolysis measured in the absence or presence of 5'-guanylyl-imidodiphosphate (Gpp(NH)p), or (at greater than or equal to 2.3 mM Li+) upon the stimulation of rat cerebral cortical inositol phospholipid breakdown by either carbachol, noradrenaline or NaF measured at either 6 or 18 mM K+. The increase in assay [K+] greatly enhanced the inositol phospholipid response to carbachol but not to NaF. The inhibitory effect of carbachol upon forskolin-stimulated adenylate cyclase was not affected by raising the [K+] from 6 to 18 mM. At 6 mM K+ (both in the absence and presence of 15 microM AlCl3), the effects of carbachol and NaF upon inositol phospholipid breakdown were essentially additive, whereas at 18 mM K+, the breakdown response to carbachol (antagonised by pirenzepine with a pA2 value of 7.6) was similar in the absence and presence of NaF. It is concluded that in the rat cerebral cortex: (a) Li+ does not affect the function of either the phosphoinositide-specific phospholipase C enzyme itself or the Gp coupled to this enzyme; (b) the difference between the additivity between NaF and carbachol seen at different assay [K+] may reflect the K(+)-dependent changes in the tetrodotoxin-resistant and tetrodotoxin-sensitive pathways of carbachol stimulation of inositol phospholipid breakdown reported by Gurwitz and Sokolovsky (1987, Biochemistry 26, 633); and (c) the effect of K+ on muscarinic receptor-coupled inositol phospholipid breakdown is not found for muscarinic receptors inhibitorily coupled to adenylate cyclase. Evidence is also presented to suggest that NaF affects the dephosphorylation of the formed [3H]inositol polyphosphates.

Differential enhancement by potassium ions of M1-type and M2-type muscarinic receptor-mediated phosphoinositide breakdown in the rat brain.

Raising the assay [K+] from 6 to 18 mM enhances the inositol phospholipid breakdown response to carbachol in rat brain miniprisms. In the frontal cortex, the degree of enhancement by K+ was independent of the carbachol concentration used, whereas in the striatum, a significantly higher degree of enhancement was seen at 1000 than at 50 microM carbachol. The carbachol-stimulated inositol phospholipid breakdown was antagonized by pirenzepine at both [K+] with potencies suggesting involvement of M1-type muscarinic receptors in the frontal cortex and both M1- and M2-type muscarinic receptors in the striatum. It is suggested that the response mediated by the M1-type receptors is enhanced to a greater degree by raised [K+] than that mediated by the M2-type receptors.

Impaired performance of rats in the Morris swim-maize test late in abstinence following long-term sodium barbital treatment.

Rats were tested for place learning in the Morris swim maze on days 110-114 of abstinence following 48 weeks of treatment with sodium barbital. A retarded acquisition of the swim-maze task, that could not be ascribed to motor impairments, was found in the barbital-treated rats. There was a significant difference in brain weight, but there were no significant differences between the control and barbital-treated rats in the frontal cortical concentrations of noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), nor in the intra- and extrasynaptosomal activities of cerebral cortical monoamine oxidase towards NA and 5-HT. Postsynaptically, neither the cerebral cortical inositol phospholipid breakdown responses to carbachol and NA (mediated by muscarinic and alpha 1-adrenergic receptors, respectively), nor the striatal and cortical densities of muscarinic receptors labelled by [3H]quinuclidinyl benzilate [( 3H]QNB) were found significantly to be altered in the barbital-treated rats. A strong correlation between the density of striatal and cortical [3H]QNB binding sites was seen for the barbital-treated (r = 0.91) but not for the control (r = -0.05) rats. It is suggested that the deficit in performance of the barbital-treated rats in the Morris maze may be related to a cholinergic dysfunction.

Stimulation of inositol phospholipid breakdown in pig brain miniprisms by carbachol and monoamines: effect of K+.

1. The effects of carbachol, monoamines and K+ upon the rate of inositol phospholipid breakdown in pig brain miniprisms have been investigated. 2. In the striatum, carbachol (EC50 approx. 1 microM) and noradrenaline (EC50 approx. 25 microM) stimulated inositol phospholipid breakdown, whereas 5-hydroxytryptamine (1-1000 microM) was without effect. 3. The rate of inositol phospholipid breakdown was increased by raising the assay [K+] to greater than or equal to 40 mM. In the hippocampus and hypothalamus, a synergistic effect between K+ and carbachol was noted, whereas in the striatum, the effects were additive. 4. In striatal and hippocampal miniprisms, dopamine also increased inositol phospholipid breakdown, albeit only at high (greater than or equal to 1 mM) concentrations. Dopamine (1 mM) reduced the stimulation produced by noradrenaline (1 mM), suggesting that the effect of dopamine is due to a weak noradrenergic action of this catecholamine.

Stimulation of inositol phospholipid breakdown in rat cortical and hippocampal miniprisms by noradrenaline, 5-hydroxytryptamine and carbachol: some methodological aspects.

After incubation of miniprisms from rat cerebral cortex or hippocampus with 3H-myo-inositol, labelling of the phospholipid ("Lipid") and inositol phosphate ("InsP") fractions was found. Inositol phospholipid hydrolysis ("PI breakdown") was stimulated by noradrenaline, 5-hydroxytryptamine and carbachol. Expressing data as InsP/(Lipid + InsP) was found to be a superior measure of the rate of PI breakdown compared with the more commonly used InsP d.p.m. unit, since the former was found to be independent of the volume of the miniprism aliquot used and the degree of labelling of inositol phospholipids. The PI breakdown responses to noradrenaline, 5-hydroxytryptamine and particularly carbachol were found to be enhanced by increasing the assay [K+] from 5.88 mM to 18.2 mM. Storage of hippocampal samples at -70 degrees by the "slow freeze-fast thaw" method of Hardy et al. (1983) resulted in a decreased degree of labelling of the Lipid and InsP fractions and a loss of the PI response to noradrenaline when assayed at a [K+] of 5.88 mM, but a reasonable response was seen in these samples at an assay [K+] of 18.2 mM. The temperature of the Krebs-Henseleit buffer used in the preparation of the miniprisms was found to be important for the PI breakdown response.

Enhancement by potassium of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms: comparison with other depolarising agents.

Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved.